Insecticidal proteins

ABSTRACT

Compositions and methods for controlling plant pests are disclosed. In particular, novel engineered hybrid insecticidal proteins (eHIPs) having toxicity to at least corn rootworm are provided. By fusing unique combinations of complete or partial variable regions and conserved blocks of at least two different Bacillus thuringiensis (Bt) Cry proteins or a modified Cry proteins an eHIP having activity against corn rootworm is designed. Nucleic acid molecules encoding the novel eHIPs are also provided. Methods of making the eHIPs and methods of using the eHIPs and nucleic acids encoding the eHIPs of the invention, for example in transgenic plants to confer protection from insect damage are also disclosed.

CROSS-REFERENCE

This application is a divisional of, and claims priority to, U.S. patentapplication Ser. No. 13/623,921, filed Sep. 21, 2012, which is adivisional of U.S. patent application Ser. No. 12/529,246, now U.S. Pat.No. 8,309,516, filed Aug. 31, 2009, which is a § 371 of PCT/US08/58182,filed Mar. 26, 2008, which claims priority to U.S. ProvisionalApplication 60/920,493, filed Mar. 28, 2007, all of which areincorporated herein by reference in their entirety.

BACKGROUND

The present invention relates to the fields of protein engineering,plant molecular biology and pest control. More particularly theinvention relates to novel engineered hybrid proteins havinginsecticidal activity, nucleic acids whose expression results in theinsecticidal proteins, and methods of making and methods of using theinsecticidal proteins and corresponding nucleic acids to controlinsects.

Insect pests are a major cause of crop losses. In the US alone, billionsof dollars are lost every year due to infestation by various genera ofinsects. In addition to losses in field crops, insect pests are also aburden to vegetable and fruit growers, to producers of ornamentalflowers, and they are a nuisance to gardeners and homeowners.

Species of corn rootworm are considered to be the most destructive cornpests. In the United States, the three important species are Diabroticavirgifera virgifera, the western corn rootworm, D. longicornis barberi,the northern corn rootworm and D. undecimpunctata howardi, the southerncorn rootworm. Only western and northern corn rootworms are consideredprimary pests of corn in the US Corn Belt. An important corn rootwormpest in the Southern US is the Mexican corn rootworm, Diabroticavirgifera zeae. Corn rootworm larvae cause the most substantial plantdamage by feeding almost exclusively on corn roots. This injury has beenshown to increase plant lodging, to reduce grain yield and vegetativeyield as well as alter the nutrient content of the grain. Larval feedingalso causes indirect effects on corn by opening avenues through theroots for bacterial and fungal infections which lead to root and stalkrot diseases. Adult corn rootworms are active in cornfields in latesummer where they feed on ears, silks and pollen, thus interfering withnormal pollination.

Corn rootworms are mainly controlled by intensive applications ofchemical pesticides, which are active through inhibition of insectgrowth, prevention of insect feeding or reproduction, or cause death.Good corn rootworm control can thus be reached, but these chemicals cansometimes also affect other, beneficial organisms. Another problemresulting from the wide use of chemical pesticides is the appearance ofresistant insect varieties. Yet another problem is due to the fact thatcorn rootworm larvae feed underground thus making it difficult to applyrescue treatments of insecticides. Therefore, most insecticideapplications are made prophylactically at the time of planting. Thispractice results in a large environmental burden. This has beenpartially alleviated by various farm management practices, but there isan increasing need for alternative pest control mechanisms.

Biological pest control agents, such as Bacillus thuringiensis (Bt)strains expressing pesticidal toxins like δ-endotoxins(delta-endotoxins; also called crystal toxins or Cry proteins), havealso been applied to crop plants with satisfactory results againstprimarily lepidopteran insect pests. The δ-endotoxins are proteins heldwithin a crystalline matrix that are known to possess insecticidalactivity when ingested by certain insects. The various δ-endotoxins havebeen classified based upon their spectrum of activity and sequencehomology. Prior to 1990, the major classes were defined by theirspectrum of activity with the Cry1 proteins active against Lepidoptera(moths and butterflies), Cry2 proteins active against both Lepidopteraand Diptera (flies and mosquitoes), Cry3 proteins active againstColeoptera (beetles) and Cry4 proteins active against Diptera (Hofte &Whitely, 1989, Microbiol. Rev. 53:242-255). A new nomenclature wasdeveloped in 1998 which systematically classifies the Cry proteins basedon amino acid sequence homology rather than insect target specificities(Crickmore et al. 1998, Microbiol. Molec. Biol. Rev. 62:807-813).

The spectrum of insecticidal activity of an individual δ-endotoxin fromBt is quite narrow, with a given δ-endotoxin being active against only afew species within an Order. For instance, a Cry3A toxin is known to bevery toxic to the Colorado potato beetle, Leptinotarsa decemlineata, buthas very little or no toxicity to related beetles in the genusDiabrotica (Johnson et al., 1993, J. Econ. Entomol. 86:330-333).According to Slaney et al. (1992, Insect Biochem. Molec. Biol. 22:9-18)a Cry3A toxin is at least 2000 times less toxic to southern cornrootworm larvae than to the Colorado potato beetle. It is also knownthat Cry3A has little or no toxicity to the western corn rootworm ornorthern corn rootworm.

Specificity of the δ-endotoxins is the result of the efficiency of thevarious steps involved in producing an active toxic protein and itssubsequent interaction with the epithelial cells in an insect mid-gut.To be insecticidal, most known δ-endotoxins must first be ingested bythe insect and proteolytically activated to form an active toxin.Activation of the insecticidal crystal (Cry) proteins is a multi-stepprocess. After ingestion, the crystals must first be solubilized in theinsect gut. Once solubilized, the δ-endotoxins are activated by specificproteolytic cleavages. The proteases in the insect gut can play a rolein specificity by determining where the δ-endotoxin is processed. Oncethe δ-endotoxin has been solubilized and processed it binds to specificreceptors on the surface of the insects' mid-gut epithelium andsubsequently integrates into the lipid bilayer of the brush bordermembrane. Ion channels then form disrupting the normal function of themidgut eventually leading to the death of the insect.

In Lepidoptera, which have alkaline pH guts, gut proteases processδ-endotoxins for example, Cry1Aa, Cry1Ab, Cry1Ac, Cry1B and Cry1F, from130-140 kDa protoxins to toxic proteins of approximately 60-70 kDa.Processing of the protoxin to toxin has been reported to proceed byremoval of both N- and C-terminal amino acids with the exact location ofprocessing being dependent on the specific δ-endotoxin and the specificinsect gut fluids involved (Ogiwara et al., 1992, J. Invert. Pathol.60:121-126). Thus activation requires that the entire C-terminalprotoxin tail region be cleaved off. This proteolytic activation of aδ-endotoxin can play a significant role in determining its specificity.

Coleopteran insects have guts that are more neutral to acidic andcoleopteran-specific δ-endotoxins are similar to the size of theactivated lepidopteran-specific toxins. Therefore, the processing ofcoleopteran-specific δ-endotoxins was formerly considered unnecessaryfor toxicity. However, data suggests that coleopteran-activeδ-endotoxins are solubilized and proteolyzed to smaller toxicpolypeptides. A 73 kDa Cry3A δ-endotoxin protein produced by B.thuringiensis var. tenebrionis is readily processed in the bacterium atthe N-terminus, losing 49-57 residues during or after crystal formationto produce the commonly isolated 67 kDa form (Carroll et al., 1989,Biochem. J. 261:99-105). McPherson et al., (1988, Biotechnology 6:61-66)also demonstrated that a native cry3A coding sequence contains twofunctional translational initiation codons in the same reading frame,one coding for a 73 kDa protein and the other coding for a 67 kDaprotein starting at Met-1 and Met-48 respectively, of the deduced aminoacid sequence. Both proteins then can be considered naturally occurringfull-length Cry3A proteins.

As more knowledge has been gained as to how the δ-endotoxins function,attempts to engineer δ-endotoxins to have new activities have increased.Engineering δ-endotoxins was made more possible by solving the threedimensional structure of Cry3A in 1991 (Li et al., 1991, Nature353:815-821). Li et al. determined that a Cry3A protein has threestructural domains: the N-terminal domain I, from residues 58-290,consists of 7 α-helices, domain II, from residues 291-500, containsthree β-sheets in a so-called Greek key-conformation, and the C-terminaldomain III, from residues 501-644, is a β-sandwich in a so-calledjellyroll conformation. The three dimensional structure for thelepidopteran active Cry1Aa toxin has also been solved (Grochulski etal., 1995, J. Mol. Biol. 254:447-464). The Cry1Aa toxin also has threedomains: the N-terminal domain I, from residues 33-253, domain II fromresidues 265-461, and domain III from residues 463-609 with anadditional outer strand in one of the β-sheets from by residues 254-264.If the Cry3A and Cry1Aa structures are projected on other Cry1sequences, domain I runs from about amino acid residue 28 to 260, domainII from about 260 to 460 and domain III from about 460 to 600. See,Nakamura et al., Agric. Biol. Chem. 54(3): 715-724 (1990); Li et al.,Nature 353: 815-821 (1991); Ge et al., J. Biol. Chem. 266(27):17954-17958 (1991); and Honee et al., Mol. Microbiol. 5(11): 2799-2806(1991); each of which are incorporated herein by reference. Thus, it isnow known that based on amino acid sequence homology, known Btδ-endotoxins have a similar three-dimensional structure comprising threedomains.

The toxin portions of Bt Cry proteins are also characterized by havingfive conserved blocks across their amino acid sequence numbered CB1 toCB5 from N-terminus to C-terminus, respectively (Hofte & Whiteley,supra). Conserved block 1 (CB1) comprises approximately 29 amino acids,conserved block 2 (CB2) comprises approximately 67 amino acids,conserved block 3 (CB3) comprises approximately 48 amino acids,conserved block 4 (CB4) comprises approximately 10 amino acids andconserved block 5 (CB5) comprises approximately 12 amino acids. Thesequences before and after these five conserved blocks are highlyvariable and thus are designated the “variable regions,” V1-V6. Domain Iof a Bt δ-endotoxin typically comprises variable region 1, conservedblock 1, variable region 2, and the N-terminal 52 amino acids ofconserved block 2. Domain II typically comprises approximately theC-terminal 15 amino acids of conserved block 2, variable region 3, andapproximately the N-terminal 10 amino acids of conserved block 3. DomainIII typically comprises approximately the C-terminal 38 amino acids ofconserved block 3, variable region 4, conserved block 4, variable region5, and conserved block 5. The Cry1 lepidopteran active toxins, amongother delta-endotoxins, have a variable region 6 with approximately 1-3amino acids lying within domain III.

Many Bt strains and δ-endotoxins are active against different insectspecies and nematodes. However, relatively few of these strains andtoxins have activity against coleopteran insects. Further, most of thenow known native coleopteran-active δ-endotoxins, for example Cry3A,Cry3B, Cry3C, Cry7A, Cry8A, Cry8B, and Cry8C, have insufficient oraltoxicity against corn rootworm to provide adequate field control ifdelivered, for example, through microbes or transgenic plants.Therefore, other approaches for producing novel toxins active againstcorn rootworm need to be explored.

Lepidopteran-active δ-endotoxins have been engineered in attempts toimprove specific activity or to broaden the spectrum of insecticidalactivity. For example, the silk moth (Bombyx mori) specificity domainfrom a Cry1Aa protein was moved to a Cry1Ac protein, thus imparting anew insecticidal activity to the resulting hybrid Bt protein (Ge et al.1989, PNAS 86: 4037-4041). Also, Bosch et al. 1998 (U.S. Pat. No.5,736,131, incorporated herein by reference) describes Bacillusthuringiensis hybrid toxins comprising at their C-terminus domain III ofa first Cry protein and at its N-terminus domains I and II of a secondCry protein. Such hybrid toxins were shown to have altered insecticidalspecificity against lepidopteran insects. For example, the H04 hybridtoxin, which is also described in De Maagd et al., Appl. Environ.Microbiol. 62(5): 1537-1543 (1996), comprises at its N-terminus domainsI and II of a Cry1Ab and at its C-terminus domain III of a Cry1C. H04 isreportedly highly toxic to the lepidopteran insect Spodoptera exigua(beet armyworm) compared with the parental Cry1Ab toxin andsignificantly more toxic than the Cry1C parental toxin. It has also beenshown that substitution of domain III of toxins, which are not activeagainst the beet armyworm such as Cry1E and Cry1Ab, by domain III ofCry1C, which is active against beet armyworm, can produce hybrid toxinsthat are active against this insect. All of the hybrids disclosed inBosch et al. use domains from lepidopteran active Cry proteins to makenew toxins with lepidopteran activity. The results do suggest thatdomain III of Cry1C is an important determinant of specificity for beetarmyworm. See also, Bosch et al., FEMS Microbiology Letters 118: 129-134(1994); Bosch et al., Bio/Technology 12: 915-918 (1994); De Maagd etal., Appl. Environ. Microbiol. 62(8): 2753-2757 (1996); and De Maagd etal., Mol. Microbiol. 31(2): 463-471 (1999); each of which isincorporated herein by reference.

Several attempts at engineering the coleopteran-active δ-endotoxins havebeen reported. Chen and Stacy (U.S. Pat. No. 7,030,295, hereinincorporated by reference) successfully created a corn rootworm activetoxin by inserting a non-naturally occurring protease recognition sitein domain I, domain III, or both domains I and III of a Cry3A protein.One of the resulting modified Cry3A proteins, designated Cry3A055,having a protease recognition site inserted in domain I, was activeagainst several species of Diabrotica. Van Rie et al., 1997, (U.S. Pat.No. 5,659,123) engineered Cry3A by randomly replacing amino acids,thought to be important in solvent accessibility, in domain II with theamino acid alanine. Several of these random replacements confined todomain II were reportedly involved in increased western corn rootwormtoxicity. However, others have shown that some alanine replacements indomain II of Cry3A result in disruption of receptor binding orstructural instability (Wu and Dean, 1996, J. Mol. Biol. 255: 628-640).English et al., 1999, (Intl. Pat. Appl. Publ. No. WO 99/31248) reportedamino acid substitutions in Cry3Bb that caused increases in toxicity tosouthern and western corn rootworm. However, of the 35 reported Cry3Bbmutants, only three, with mutations primarily in domain II and thedomain I-domain II interface, were active against western corn rootworm.Further, the variation in toxicity of wild-type Cry3Bb against westerncorn rootworm in the same assays appear to be greater than any of thedifferences between the mutated Cry3Bb toxins and the wild-type Cry3Bb.Shadenkov et al. (1993, Mol. Biol. 27:586-591), made a hybrid protein byfusing amino acids 48-565 of a Cry3A protein to amino acids 526-725 of aCry1Aa protein. Therefore, the cross-over between Cry3A and Cry1Aasequence was in conserved block 4 located in domain III. Cry3A is veryactive against the Colorado potato beetle (Leptinotarsa decemlineata).However, the hybrid protein disclosed by Shadenkov et al. was not activeagainst Colorado potato beetle even though more than 75% of the hybridprotein was made up of Cry3A sequence. Thus, the addition of only 25% ofCry1Aa sequence destroyed activity against a coleopteran insect that theparent Cry3A was active against. This suggests that hybrid proteins madeby fusing portions of a coleopteran-active Cry protein, e.g. Cry3A, anda lepidopteran-active Cry protein, e.g. Cry1A, would not have activityagainst coleopteran insects, particularly a coleopteran insect that isnot naturally susceptible to Cry3A like corn rootworm.

In view of the above discussion, there remains a need to design new andeffective pest control agents that provide an economic benefit tofarmers and that are environmentally acceptable. Particularly needed areproteins that are toxic to Diabrotica species, a major pest of corn,that have a different mode of action than existing insect controlproducts as a way to mitigate the development of resistance.Furthermore, delivery of control agents through products that minimizethe burden on the environment, as through transgenic plants, aredesirable.

SUMMARY

In view of these needs, it is an object of the present invention toprovide novel engineered hybrid insecticidal proteins (eHIPs). Suchnovel eHIPs are made by fusing unique combinations of variable regionsand conserved blocks of at least two different Cry proteins andoptionally including a protoxin tail region from a Bt Cry protein at theC-terminus or an N-terminal peptidyl fragment or both. For example,without limitation, by combining complete or partial variable regionsand conserved blocks from a first Cry protein that has coleopteranactivity with complete or partial variable regions and conserved blocksfrom a second Cry protein that has lepidopteran activity, and isdifferent from the first Cry protein, and optionally including aprotoxin tail region from a lepidopteran active Bt Cry protein, or anN-terminal peptidyl fragment or both, new engineered hybrid insecticidalproteins are created that have activity against a spectrum of insectsdifferent from the first or second parent Cry proteins or both. Suchnovel eHIPs may comprise complete or partial variable regions, conservedblocks or domains from a modified Cry3A protein and a Cry proteindifferent from the modified Cry3A protein. The peptidyl fragment mayconfer insecticidal activity upon the eHIP, or may increase theinsecticidal activity of the eHIP over an eHIP without the peptidylfragment, or make the eHIP more stable than an eHIP without the peptidylfragment. The eHIPs of the invention have surprising and unexpectedtoxicity to corn rootworm (Diabrotica sp.). The invention is furtherdrawn to nucleic acids that encode the eHIPs or which is complementaryto one which hybridizes under stringent conditions with the recombinanthybrid nucleic acids according to the invention.

Also included in the invention are vectors containing such recombinant(or complementary thereto) nucleic acids; a plant or micro-organismwhich includes, and enables expression of such nucleic acids; plantstransformed with such nucleic acids, for example transgenic corn plants;the progeny of such plants which contain the nucleic acids stablyincorporated and hereditable in a Mendelian manner, and/or the seeds ofsuch plants and such progeny.

The invention also includes compositions and formulations containing theeHIPs, which are capable of inhibiting the ability of insect pests tosurvive, grow and reproduce, or of limiting insect-related damage orloss to crop plants, for example applying the eHIPs or compositions orformulations to insect-infested areas, or to prophylactically treatinsect-susceptible areas or plants to confer protection against theinsect pests.

The invention is further drawn to a method of making the eHIPs and tomethods of using the nucleic acids, for example in microorganisms tocontrol insects or in transgenic plants to confer protection from insectdamage.

The novel eHIPs described herein are highly active against insects. Forexample, the eHIPs of the present invention can be used to controleconomically important insect pests such as western corn rootworm(Diabrotica virgifera virgifera) northern corn rootworm (D. longicornisbarberi) and Mexican corn rootworm (D. virgifera zeae). Certain eHIPsmay also be used to control European corn borer (Ostrinia nubilalis) andother lepidopteran insects. The eHIPs can be used singly or incombination with other insect control strategies to confer maximal pestcontrol efficiency with minimal environmental impact.

Other aspects and advantages of the present invention will becomeapparent to those skilled in the art from a study of the followingdescription of the invention and non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1A-1E shows a sequence alignment of some eHIP embodiments withparental Cry proteins or modified Cry 3A used to construct the eHIPs,including, a Cry3A, Cry1Ab, and Cry3A055, and indicates percentidentity. N-terminal peptidyl fragments are underlined. The 5 conservedblocks are labeled CB1-CB5. Location of junctions between domains I, IIand III are designated by a vertical dashed line. A cathepsin G proteaserecognition sequence, AAPF, is in bold.

FIG. 2A-2E shows an alignment of eHIP embodiments that are activeagainst at least western corn rootworm and indicates percent identitycompared to the 8AF eHIP. N-terminal peptidyl fragments are singleunderlined. C-terminal protoxin tail regions are double underlined. The5 conserved blocks are labeled CB1-CB5. Locations of junctions betweendomains I, II and III are indicated by “↓” and labeled accordingly.Locations of crossover positions are indicted by a “♦”. A cathepsin Gprotease recognition sequence, AAPF, is in bold.

FIG. 3 shows a map of recombinant vector 12207 used to transform corncomprising an expression cassette with a maize ubiquitin promoteroperably linked to a FRCG coding sequence operably linked to a NOSterminator.

FIG. 4 shows a map of recombinant vector 12161 used to transform corncomprising an expression cassette with a maize ubiquitin promoteroperably linked to a FR8a coding sequence operably linked to a NOSterminator.

FIG. 5 shows a map of recombinant vector 12208 used to transform corncomprising an expression cassette with a cestrum yellow leaf curlingvirus promoter (cmp) operably linked to a FRCG coding sequence operablylinked to a NOS terminator.

FIG. 6 shows a map of recombinant vector 12274 used to transform corncomprising an expression cassette with a cestrum yellow leaf curlingvirus promoter (cmp) operably linked to a FR8a coding sequence operablylinked to a NOS terminator.

FIG. 7 shows a map of recombinant vector 12473 used to transform corncomprising an expression cassette with a maize ubiquitin promoter (ubi)operably linked to a FRD3 coding sequence operably linked to a NOSterminator.

FIG. 8 shows a map of recombinant vector 12474 used to transform corncomprising an expression cassette with a cestrum yellow leaf curlingvirus promoter (cmp) operably linked to a FRD3 coding sequence operablylinked to a NOS terminator.

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the 2OL-8a nucleotide sequence.

SEQ ID NO: 2 is the 2OL-8a encoded by SEQ ID NO: 1.

SEQ ID NO: 3 is the FR8a nucleotide sequence.

SEQ ID NO: 4 is the FR8a encoded by SEQ ID NO: 3.

SEQ ID NO: 5 is the FRCG nucleotide sequence.

SEQ ID NO: 6 is the FRCG encoded by SEQ ID NO: 5.

SEQ ID NO: 7 is the FR8a-9F nucleotide sequence.

SEQ ID NO: 8 is the FR8a-9F encoded by SEQ ID NO: 7.

SEQ ID NO: 9 is the FR-9F-catg nucleotide sequence.

SEQ ID NO: 10 is the FR-9F-catg encoded by SEQ ID NO: 9.

SEQ ID NO: 11 is the FR8a-12AA nucleotide sequence.

SEQ ID NO: 12 is the FR8a-12AA encoded by SEQ ID NO: 11.

SEQ ID NO: 13 is the WR-9mut nucleotide sequence.

SEQ ID NO: 14 is the WR-9mut encoded by SEQ ID NO: 13.

SEQ ID NO: 15 is the FRD3 nucleotide sequence.

SEQ ID NO: 16 is the FRD3 encoded by SEQ ID NO: 15.

SEQ ID NO: 17 is the FR-12-cg-dm3 nucleotide sequence.

SEQ ID NO: 18 is the FR-12-cg-dm3 encoded by SEQ ID NO: 17.

SEQ ID NO: 19 is the 9F-cg-del6 nucleotide sequence.

SEQ ID NO: 20 is the 9F-cg-del6 encoded by SEQ ID NO: 19.

SEQ ID NO: 21 is the FR-cg-dm3 nucleotide sequence.

SEQ ID NO: 22 is the FR-cg-dm3 encoded by SEQ ID NO: 21.

SEQ ID NO: 23 is the 9F-cg-dm3 nucleotide sequence.

SEQ ID NO: 24 is the 9F-cg-dm3 encoded by SEQ ID NO:23.

SEQ ID NO: 25 is the B8a nucleotide sequence.

SEQ ID NO: 26 is the B8a encoded by SEQ ID NO: 25.

SEQ ID NO: 27 is the 5*B8a nucleotide sequence.

SEQ ID NO: 28 is the 5*B8a encoded by SEQ ID NO: 27.

SEQ ID NO: 29 is the V3A nucleotide sequence.

SEQ ID NO: 30 is the V3A encoded by SEQ ID NO: 29.

SEQ ID NO: 31 is the V4F nucleotide sequence.

SEQ ID NO: 32 is the V4F encoded by SEQ ID NO: 31.

SEQ ID NO: 33 is the 5*V4F nucleotide sequence.

SEQ ID NO: 34 is the 5*V4F encoded by SEQ ID NO: 33.

SEQ ID NO: 35 is the 2OL-7 nucleotide sequence.

SEQ ID NO: 36 is the 2OL-7 encoded by SEQ ID NO: 35.

SEQ ID NO: 37 is the T7-2OL-7 nucleotide sequence.

SEQ ID NO: 38 is the T7-2OL-7 encoded by SEQ ID NO: 37.

SEQ ID NO: 39 is the 5*2OL-7 nucleotide sequence.

SEQ ID NO: 40 is the 5*2OL-7 encoded by SEQ ID NO: 39.

SEQ ID NO: 41 is the 2OL-10 nucleotide sequence.

SEQ ID NO: 42 is the 2OL-10 encoded by SEQ ID NO: 41.

SEQ ID NO: 43 is the 5*2OL-10 nucleotide sequence.

SEQ ID NO: 44 is the 5*2OL-10 encoded by SEQ ID NO: 43.

SEQ ID NO: 45 is the 2OL-12A nucleotide sequence.

SEQ ID NO: 46 is the 2OL-12A encoded by SEQ ID NO: 45.

SEQ ID NO: 47 is the 2OL-13 nucleotide sequence.

SEQ ID NO: 48 is the 201-13 encoded by SEQ ID NO: 47.

SEQ ID NO: 49 is the V5&6 nucleotide sequence.

SEQ ID NO: 50 is the V5&6 encoded by SEQ ID NO: 49.

SEQ ID NO: 51 is the 5*V5&6 nucleotide sequence.

SEQ ID NO: 52 is the 5*V5&6 encoded by SEQ ID NO: 51.

SEQ ID NO: 53 is the 88A-dm3 nucleotide sequence.

SEQ ID NO: 54 is the 88A-dm3 encoded by SEQ ID NO: 53.

SEQ ID NO: 55 is the FR(1Fa) nucleotide sequence.

SEQ ID NO: 56 is the FR(1Fa) encoded by SEQ ID NO: 55.

SEQ ID NO: 57 is the FR(1Ac) nucleotide sequence.

SEQ ID NO: 58 is the FR(1Ac) encoded by SEQ ID NO: 57.

SEQ ID NO: 59 is the FR(1Ia) nucleotide sequence.

SEQ ID NO: 60 is the FR(1Ia) encoded by SEQ ID NO: 59.

SEQ ID NO: 61 is the DM23A nucleotide sequence.

SEQ ID NO: 62 is the DM23A encoded by SEQ ID NO: 61.

SEQ ID NO: 63 is the 8AF nucleotide sequence.

SEQ ID NO: 64 is the 8AF encoded by SEQ ID NO: 63.

SEQ ID NO: 65 is the 5*cry3A055 nucleotide sequence.

SEQ ID NO: 66 is the 5*Cry3A055 encoded by SEQ ID NO: 65.

SEQ ID NO: 67 is a maize optimized cry3A nucleotide sequence.

SEQ ID NO: 68 is the Cry3A encoded by SEQ ID NO: 67.

SEQ ID NO: 69 is the cry3A055 nucleotide sequence.

SEQ ID NO: 70 is the Cry3A055 encoded by SEQ ID NO: 69.

SEQ ID NO: 71 is a maize optimized cry1Ab nucleotide sequence.

SEQ ID NO: 72 is the Cry1Ab encoded by SEQ ID NO: 71.

SEQ ID NO: 73 is a maize optimized cry1Ba nucleotide sequence.

SEQ ID NO: 74 is the Cry1Ba encoded by SEQ ID NO: 73.

SEQ ID NO: 75 is a maize optimized cry1Fa nucleotide sequence.

SEQ ID NO: 76 is the Cry1Fa encoded by SEQ ID NO: 75.

SEQ ID NO: 77 is a cry8Aa nucleotide sequence.

SEQ ID NO: 78 is the Cry8Aa encoded by SEQ ID NO: 77.

SEQ ID NO: 79 is a cry1Ac nucleotide sequence.

SEQ ID NO: 80 is the Cry1Ac encoded by SEQ ID NO: 79.

SEQ ID NO: 81 is a cry1Ia nucleotide sequence.

SEQ ID NO: 82 is the Cry1Ia encoded by SEQ ID NO: 81.

SEQ ID NOs 83-125 are primer sequences useful in the present invention.

SEQ ID NOs 126-134 are N-terminal peptidyl fragments.

SEQ ID NO: 135 is a full-length Cry3A protein.

SEQ ID NO: 136-143 are primer sequences useful in the present invention.

SEQ ID NO: 144 is the T7-8AF coding sequence.

SEQ ID NO: 145 is the T7-8AF encoded by ASEQ ID NO: 144.

SEQ ID NO: 146 is the −catG8AF coding sequence.

SEQ ID NO: 147 is the −CatG8AF encoded by SEQ ID NO: 146.

SEQ ID NO: 148 is the 8AFdm3 coding sequence.

SEQ ID NO: 149 is the 8AFdm3 encoded by SEQ ID NO: 148.

SEQ ID NO: 150 is the 8AFlongdm3 coding sequence.

SEQ ID NO: 151 is the 8AFlongdm3 encoded by SEQ ID NO: 150.

SEQ ID NO: 152 is the cap8AFdm3 coding sequence.

SEQ ID NO: 153 is the cap8AFdm3 encoded by SEQ ID NO: 152.

SEQ ID NO: 154 is the 8AFdm3T coding sequence.

SEQ ID NO: 155 is the 8AFdm3T encoded by SEQ ID NO: 154.

SEQ ID NO: 156 is the 8AFlongdm3T coding sequence.

SEQ ID NO: 157 is the 8AFlongdm3T encoded by SEQ ID NO: 156.

SEQ ID NO: 158 is the cap8AFdm3T coding sequence.

SEQ ID NO: 159 is the cap8AFdm3T encoded by SEQ ID NO: 158.

SEQ ID NO: 160 is the FR8a+34 eHIP.

DEFINITIONS

For clarity, certain terms used in the specification are defined andpresented as follows:

“Activity” of the eHIPs of the invention is meant that the eHIPsfunction as orally active insect control agents, have a toxic effect, orare able to disrupt or deter insect feeding, which may or may not causedeath of the insect. When an eHIP of the invention is delivered to theinsect, the result is typically death of the insect, or the insect doesnot feed upon the source that makes the eHIP available to the insect.

“Associated with/operatively linked” refer to two nucleic acids that arerelated physically or functionally. For example, a promoter orregulatory DNA sequence is said to be “associated with” a DNA sequencethat codes for RNA or a protein if the two sequences are operativelylinked, or situated such that the regulatory DNA sequence will affectthe expression level of the coding or structural DNA sequence.

In the context of the present invention, a “chimeric insecticidalprotein” (CIP) is an insecticidal protein comprising a peptidyl fragmentfused to the N-terminus of an eHIP. The peptidyl fragment may conferinsecticidal activity upon the eHIP, may increase the insecticidalactivity of the eHIP over an eHIP without the peptidyl fragment, or maymake the eHIP more stable than an eHIP without the peptidyl fragment,particularly against at least western corn rootworm. The peptidylfragment is an amino acid sequence that is typically heterologus to (notderived from) a Bt Cry protein but may be derived from a Bt Cry protein.Such peptidyl fragments extend from the N-terminus of the insecticidalprotein and do not naturally occur at the N-terminus of Bt Cry proteins.One example of an N-terminal peptidyl fragment has the amino acidsequence MTSNGRQCAGIRP (SEQ ID NO: 129) which is not derived from a BtCry protein.

A “coding sequence” is a nucleic acid sequence that is transcribed intoRNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.Preferably the RNA is then translated in an organism to produce aprotein.

In the context of the present invention, “connecting” nucleic acidsmeans to join two or more nucleic acids together using any means knownin the art. For example, without limitation, the nucleic acids may beligated together using for example, DNA ligase, or may be annealed usingPCR. The nucleic acids may also be joined by chemically synthesizing anucleic acid using the sequence of two or more separate nucleic acids.

To “control” insects means to inhibit, through a toxic effect, theability of insect pests to survive, grow, feed, and/or reproduce, or tolimit insect-related damage or loss in crop plants. To “control” insectsmay or may not mean killing the insects, although it preferably meanskilling the insects.

In the context of the present invention, “corresponding to” means thatwhen the amino acid sequences of certain proteins (for example Bt Cryproteins or modified Cry3A proteins) are aligned with each other, theamino acids that align with certain enumerated positions in for example,but not limited to, a Cry3A toxin (either SEQ ID NO: 68 or SEQ ID NO:134); a Cry3A055 toxin (SEQ ID NO: 70); or a Cry1Ab toxin (SEQ ID NO:72), but that are not necessarily in these exact numerical positionsrelative to the reference amino acid sequence, particularly as itrelates to identification of domains I, II and III, and/or the conservedblocks and variable regions, these amino acid positions “correspond to”each other. For example, in delineating Domain I of a hybrid protein,amino acids 11-244 of a Cry3A055 protein (SEQ ID NO: 70) correspond toamino acids 58-290 of a native Cry3A toxin (SEQ ID NO: 135) or to aminoacids 11-243 of a native Cry3A toxin (SEQ ID NO: 68) or to amino acids33-254 of a native Cry1Ab toxin.

In the context of the present invention the words “Cry protein” can beused interchangeably with the words “delta-endotoxin” or “δ-endotoxin.”

In the context of the present invention, an “engineered hybridinsecticidal protein” (eHIP) is an insecticidal protein created byfusing unique combinations of variable regions and conserved blocks ofat least two different Cry proteins. Such novel eHIPs may comprisecomplete or partial variable regions, conserved blocks or domains from amodified Cry3A protein and a Cry protein different from the modifiedCry3A protein. The eHIPs of the invention may optionally include aprotoxin tail region from a Bt Cry protein or an N-terminal peptidylfragment or both. For example without limitation, an eHIP is created bycombining in an N-terminal to C-terminal direction, amino acids 1-468 ofa Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1,conserved block 1, variable region 2, conserved block 2, variable region3, and the N-terminal 24 amino acids of conserved block 3, and aminoacids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 24 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5 and variableregion 6, and a 38 amino acid region of a Cry1Ab protoxin tail. An eHIPthat comprises an N-terminal peptidyl fragment may also be designated asa “chimeric insecticidal protein (CIP).”

To “deliver” an eHIP means that the eHIP comes in contact with aninsect, resulting in a toxic effect and control of the insect. The eHIPmay be delivered in many recognized ways, e.g., through a transgenicplant expressing the eHIP, formulated protein composition(s), sprayableprotein composition(s), a bait matrix, or any other art-recognized toxindelivery system.

“Effective insect-controlling amount” means that concentration of aneHIP that inhibits, through a toxic effect, the ability of insects tosurvive, grow, feed and/or reproduce, or to limit insect-related damageor loss in crop plants. “Effective insect-controlling amount” may or maynot mean killing the insects, although it preferably means killing theinsects.

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The expression cassettecomprising the nucleotide sequence of interest may have at least one ofits components heterologous with respect to at least one of its othercomponents. The expression cassette may also be one that is naturallyoccurring but has been obtained in a recombinant form useful forheterologous expression. Typically, however, the expression cassette isheterologous with respect to the host, i.e., the particular nucleic acidsequence of the expression cassette does not occur naturally in the hostcell and must have been introduced into the host cell or an ancestor ofthe host cell by a transformation event. The expression of thenucleotide sequence in the expression cassette may be under the controlof a constitutive promoter or of an inducible promoter that initiatestranscription only when the host cell is exposed to some particularexternal stimulus. In the case of a multicellular organism, such as aplant, the promoter can also be specific to a particular tissue, ororgan, or stage of development.

A “gene” is a defined region that is located within a genome and that,besides the aforementioned coding nucleic acid sequence, comprisesother, primarily regulatory, nucleic acids responsible for the controlof the expression, that is to say the transcription and translation, ofthe coding portion. A gene may also comprise other 5′ and 3′untranslated sequences and termination sequences. Further elements thatmay be present are, for example, introns. The regulatory nucleic acidsequence of the gene may not normally be operatively linked to theassociated nucleic acid sequence as found in nature and thus would be achimeric gene.

“Gene of interest” refers to any gene which, when transferred to aplant, confers upon the plant a desired characteristic such asantibiotic resistance, virus resistance, insect resistance, diseaseresistance, or resistance to other pests, herbicide tolerance, improvednutritional value, improved performance in an industrial process oraltered reproductive capability. The “gene of interest” may also be onethat is transferred to plants for the production of commerciallyvaluable enzymes or metabolites in the plant.

A “heterologous” nucleic acid sequence is a nucleic acid sequence notnaturally associated with a host cell into which it is introduced,including non-naturally occurring multiple copies of a naturallyoccurring nucleic acid sequence. A heterologous amino acid sequence isone that is not naturally associated with a native amino acid sequence,for example an amino acid sequence of a Cry protein.

A “homologous” nucleic acid sequence is a nucleic acid sequencenaturally associated with a host cell into which it is introduced.

“Homologous recombination” is the reciprocal exchange of nucleic acidfragments between homologous nucleic acid molecules.

“Identity” or “percent identity” refers to the degree of similaritybetween two nucleic acid or protein sequences. For sequence comparison,typically one sequence acts as a reference sequence to which testsequences are compared. When using a sequence comparison algorithm, testand reference sequences are input into a computer, subsequencecoordinates are designated if necessary, and sequence algorithm programparameters are designated. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated programparameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch,J. Mol. Biol. 48: 443 (1970), by the search for similarity method ofPearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988), bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally, Ausubel et al., infra).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215: 403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., 1990). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when the cumulative alignment score falls off bythe quantity X from its maximum achieved value, the cumulative scoregoes to zero or below due to the accumulation of one or morenegative-scoring residue alignments, or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a wordlength (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci.USA 89: 10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90: 5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a test nucleicacid sequence is considered similar to a reference sequence if thesmallest sum probability in a comparison of the test nucleic acidsequence to the reference nucleic acid sequence is less than about 0.1,more preferably less than about 0.01, and most preferably less thanabout 0.001.

Another widely used and accepted computer program for performingsequence alignments is CLUSTALW v1.6 (Thompson, et al. Nuc. Acids Res.,22: 4673-4680, 1994). The number of matching bases or amino acids isdivided by the total number of bases or amino acids, and multiplied by100 to obtain a percent identity. For example, if two 580 base pairsequences had 145 matched bases, they would be 25 percent identical. Ifthe two compared sequences are of different lengths, the number ofmatches is divided by the shorter of the two lengths. For example, ifthere were 100 matched amino acids between a 200 and a 400 amino acidproteins, they are 50 percent identical with respect to the shortersequence. If the shorter sequence is less than 150 bases or 50 aminoacids in length, the number of matches are divided by 150 (for nucleicacid bases) or 50 (for amino acids), and multiplied by 100 to obtain apercent identity.

Another indication that two nucleic acids are substantially identical isthat the two molecules hybridize to each other under stringentconditions. The phrase “hybridizing specifically to” refers to thebinding, duplexing, or hybridizing of a molecule only to a particularnucleotide sequence under stringent conditions when that sequence ispresent in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s)substantially” refers to complementary hybridization between a probenucleic acid and a target nucleic acid and embraces minor mismatchesthat can be accommodated by reducing the stringency of the hybridizationmedia to achieve the desired detection of the target nucleic acidsequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen (1993) LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization withNucleic Acid Probes part I chapter 2 “Overview of principles ofhybridization and the strategy of nucleic acid probe assays” Elsevier,New York. Generally, highly stringent hybridization and wash conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.Typically, under “stringent conditions” a probe will hybridize to itstarget subsequence, but to no other sequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor hybridization of complementary nucleic acids which have more than100 complementary residues on a filter in a Southern or northern blot is50% formamide with 1 mg of heparin at 42° C., with the hybridizationbeing carried out overnight. An example of highly stringent washconditions is 0.1 5M NaCl at 72° C. for about 15 minutes. An example ofstringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes(see, Sambrook, infra, for a description of SSC buffer). Often, a highstringency wash is preceded by a low stringency wash to removebackground probe signal. An example medium stringency wash for a duplexof, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes.An example low stringency wash for a duplex of, e.g., more than 100nucleotides, is 4-6×SSC at 40° C. for 15 minutes. For short probes(e.g., about 10 to 50 nucleotides), stringent conditions typicallyinvolve salt concentrations of less than about 1.0 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3, and the temperature is typically at least about 30° C. Stringentconditions can also be achieved with the addition of destabilizingagents such as formamide. In general, a signal to noise ratio of 2× (orhigher) than that observed for an unrelated probe in the particularhybridization assay indicates detection of a specific hybridization.Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the proteins that theyencode are substantially identical. This occurs, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone homologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

A further indication that two nucleic acids or proteins aresubstantially identical is that the protein encoded by the first nucleicacid is immunologically cross reactive with, or specifically binds to,the protein encoded by the second nucleic acid. Thus, a protein istypically substantially identical to a second protein, for example,where the two proteins differ only by conservative substitutions.

“Insecticidal” is defined as a toxic biological activity capable ofcontrolling insects, preferably by killing them.

A nucleic acid sequence is “isocoding with” a reference nucleic acidsequence when the nucleic acid sequence encodes a polypeptide having thesame amino acid sequence as the polypeptide encoded by the referencenucleic acid sequence.

An “isolated” nucleic acid molecule or an isolated toxin is a nucleicacid molecule or toxin that, by the hand of man, exists apart from itsnative environment and is therefore not a product of nature. An isolatednucleic acid molecule or toxin may exist in a purified form or may existin a non-native environment such as, for example without limitation, arecombinant microbial cell, plant cell, plant tissue, or plant.

A “modified Cry3A toxin” or “Cry3A055” of this invention refers to aCry3A-derived toxin having at least one additional protease recognitionsite that is recognized by a gut protease of a target insect, which doesnot naturally occur in a Cry3A toxin, as described in U.S. Pat. No.7,030,295, herein incorporated by reference.

A “modified cry3A coding sequence” according to this invention can bederived from a native cry3A coding sequence or from a synthetic cry3Acoding sequence and comprises the coding sequence of at least oneadditional protease recognition site that does not naturally occur in anunmodified cry3A gene.

A “nucleic acid molecule” or “nucleic acid sequence” is a segment ofsingle- or double-stranded DNA or RNA that can be isolated from anysource. In the context of the present invention, the nucleic acidmolecule is typically a segment of DNA.

A “plant” is any plant at any stage of development, particularly a seedplant.

A “plant cell” is a structural and physiological unit of a plant,comprising a protoplast and a cell wall. The plant cell may be in theform of an isolated single cell or a cultured cell, or as a part of ahigher organized unit such as, for example, plant tissue, a plant organ,or a whole plant.

“Plant cell culture” means cultures of plant units such as, for example,protoplasts, cell culture cells, cells in plant tissues, pollen, pollentubes, ovules, embryo sacs, zygotes and embryos at various stages ofdevelopment.

“Plant material” refers to leaves, stems, roots, flowers or flowerparts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell ortissue cultures, or any other part or product of a plant.

A “plant organ” is a distinct and visibly structured and differentiatedpart of a plant such as a root, stem, leaf, flower bud, or embryo.

“Plant tissue” as used herein means a group of plant cells organizedinto a structural and functional unit. Any tissue of a plant in plantaor in culture is included. This term includes, but is not limited to,whole plants, plant organs, plant seeds, tissue culture and any groupsof plant cells organized into structural and/or functional units. Theuse of this term in conjunction with, or in the absence of, any specifictype of plant tissue as listed above or otherwise embraced by thisdefinition is not intended to be exclusive of any other type of planttissue.

A “promoter” is an untranslated DNA sequence upstream of the codingregion that contains the binding site for RNA polymerase and initiatestranscription of the DNA. The promoter region may also include otherelements that act as regulators of gene expression.

“Regulatory elements” refer to sequences involved in controlling theexpression of a nucleotide sequence. Regulatory elements comprise apromoter operably linked to the nucleotide sequence of interest andtermination signals. They also typically encompass sequences requiredfor proper translation of the nucleotide sequence.

“Transformation” is a process for introducing heterologous nucleic acidinto a host cell or organism. In particular, “transformation” means thestable integration of a DNA molecule into the genome of an organism ofinterest.

“Transformed/transgenic/recombinant” refer to a host organism such as abacterium or a plant into which a heterologous nucleic acid molecule hasbeen introduced. The nucleic acid molecule can be stably integrated intothe genome of the host or the nucleic acid molecule can also be presentas an extrachromosomal molecule. Such an extrachromosomal molecule canbe auto-replicating. Transformed cells, tissues, or plants areunderstood to encompass not only the end product of a transformationprocess, but also transgenic progeny thereof. A “non-transformed”,“non-transgenic”, or “non-recombinant” host refers to a wild-typeorganism, e.g., a bacterium or plant, which does not contain theheterologous nucleic acid molecule.

Nucleotides are indicated by their bases by the following standardabbreviations: adenine (A), cytosine (C), thymine (T), and guanine (G).Amino acids are likewise indicated by the following standardabbreviations: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N),aspartic acid (Asp; D), cysteine (Cys; C), glutamine (Gln; Q), glutamicacid (Glu; E), glycine (Gly; G), histidine (His; H), isoleucine (Ile;1), leucine (Leu; L), lysine (Lys; K), methionine (Met; M),phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine(Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

DESCRIPTION

This invention relates to novel engineered hybrid insecticidal proteins(eHIPS), created to have activity against at least western cornrootworm, and may further include northern corn rootworm, Mexican cornrootworm, and/or Colorado potato beetle. Some eHIPs have activityagainst the lepidopteran pest, European corn borer. Such novel eHIPs aremade by fusing unique combinations of complete or partial variableregions and conserved blocks of at least two different Cry proteins andoptionally include a protoxin tail region from a Bt Cry protein at theC-terminus or an N-terminal peptidyl fragment or both. For example,without limitation, by combining complete or partial variable regionsand conserved blocks from a first Cry protein that has coleopteranactivity with complete or partial variable regions and conserved blocksfrom a second Cry protein that has lepidopteran activity and isdifferent from the first Bt Cry protein, and optionally including aprotoxin tail region from a Bt Cry protein at the C-terminus or anN-terminal peptidyl fragment or both, new engineered hybrid insecticidalproteins that have activity against a spectrum of insects that isdifferent from the first or second parent Cry protein, or both, iscreated. Such novel eHIPs may also comprise complete or partial variableregions, conserved blocks or domains from a modified Cry3A protein and aCry protein different from the modified Cry3A protein. The N-terminalpeptidyl fragment or protoxin tail region may confer insecticidalactivity upon the eHIP, may increase the insecticidal activity of aneHIP over an eHIP without the peptidyl fragment or protoxin tail region,and/or may make the eHIP more stable than an eHIP without the peptidylfragment or protoxin tail region, particularly against at least westerncorn rootworm. The amino acid sequence of the peptidyl fragmenttypically is heterologous to (i.e. not derived from) a Bt Cry protein.However, based on the teaching disclosed herein, the skilled person willrecognize that an N-terminal peptidyl fragment may be generated using anamino acid sequence derived from a Bt Cry protein. The eHIPs of theinvention have surprising and unexpected toxicity to corn rootworm,particularly to western, northern and Mexican corn rootworm. The presentinvention also relates to nucleic acids whose expression results ineHIPs, and to the making and using of the eHIPs to control insect pests.The expression of the nucleic acids results in eHIPs that can be used tocontrol coleopteran insects such as western, northern or Mexican cornrootworm, or used to control lepidopteran insects such as European cornborer, particularly when expressed in a transgenic plant such as atransgenic corn plant.

In one embodiment, the invention encompasses an engineered hybridinsecticidal protein comprising an amino acid sequence from a firstBacillus thuringiensis (Bt) Cry protein comprising complete or partialvariable regions and conserved blocks of the first Cry protein fused toan amino acid sequence from a second Bt Cry protein different from thefirst Bt Cry protein comprising complete or partial variable regions andconserved blocks of the second Cry protein, and optionally comprising:(a) a protoxin tail region of a Bt Cry protein located at theC-terminus; or (b) an N-terminal peptidyl fragment; or both (a) and (b),wherein the eHIP has activity against at least western corn rootworm.

In another embodiment, the present invention encompasses an eHIPcomprising an N-terminal region of a first Bt Cry protein fused to aC-terminal region of a second Bt Cry protein different from the first BtCry protein, wherein at least one crossover position between the firstand the second Bt Cry protein is located in conserved block 2, conservedblock 3, variable region 4 or conserved block 4, and optionallycomprising: (a) a protoxin tail region of a Bt Cry protein located atthe C-terminus; or (b) an N-terminal peptidyl fragment; or both (a) and(b), wherein the eHIP has insecticidal activity against at least westerncorn rootworm.

In another embodiment, an eHIP according to the invention comprises fromN-terminus to C-terminus variable region 1 or a C-terminal portion ofvariable region 1, conserved block 1, variable region 2, conserved block2, variable region 3, and an N-terminal portion of conserved block 3 ofa first Bt Cry protein fused to a C-terminal portion of conserved block3, variable region 4, conserved block 4, variable region 5, conservedblock 5, and variable region 6 of a second Bt Cry protein.

In another embodiment, an eHIP of the invention comprises at least twocrossover positions between an amino acid sequence from the first Bt Cryprotein and an amino acid sequence from the second Bt Cry protein. Inone embodiment, a first crossover position is located in conserved block2 and a second crossover position is located in conserved block 3. Inanother embodiment, a first crossover junction is located in conservedblock 3 and a second crossover position is located in conserved block 4.

In another embodiment, an eHIP of the invention comprises at theC-terminus a protoxin tail region of a Bt Cry protein. The protoxin tailregion may confer insecticidal activity upon the eHIP, meaning thatwithout the protoxin tail region the eHIP would not be active, mayincrease activity of the eHIP over an eHIP without the protoxin tailregion, or may make the eHIP more stable than an eHIP without theprotoxin tail region. In one embodiment, the protoxin tail region isfrom a lepidopteran active Bt Cry protein. In another embodiment, theprotoxin tail region is from a Cry1A protein. In yet another embodiment,the protoxin tail region is from a Cry1Aa or a Cry1Ab protein. Theprotoxin tail region of the invention may comprise an entire protoxintail of a Bt Cry protein or any fragment thereof. In one aspect of thisembodiment, the protoxin tail region of an eHIP comprises at least 38amino acids from the N-terminus of a protoxin tail of a Cry1Ab protein.In another aspect of this embodiment, the protoxin tail region comprisesan amino acid sequence corresponding to amino acids 611-648 of SEQ IDNO: 72. In still another aspect of this embodiment, the protoxin tailregion comprises amino acids 611-648 of SEQ ID NO: 72.

In still another embodiment, an eHIP comprises an N-terminal peptidylfragment. The N-terminal peptidyl fragment may confer insecticidalactivity upon the eHIP, meaning that without the N-terminal peptidylfragment the protein does not have insecticidal activity, or theN-terminal peptidyl fragment may increase the insecticidal activity ofthe eHIP over an eHIP without the N-terminal peptidyl fragment, or theN-terminal peptidyl fragment may make the eHIP more stable than an eHIPwithout an N-terminal peptidyl fragment. In one aspect of thisembodiment, the peptidyl fragment comprises an amino acid sequence thatis heterologous to (i.e. not derived from) a Bt Cry protein. In anotheraspect of this embodiment, the N-terminal peptidyl fragment comprises atleast 9 amino acids. In yet another aspect of this embodiment, thepeptidyl fragment comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 70, 80, 90 or 100 amino acids. In another aspectof this embodiment, the peptidyl fragment comprises greater than 100amino acids. In still another aspect of this embodiment, the N-terminalpeptidyl fragment comprises the amino acid sequence YDGRQQHRG (SEQ IDNO: 133) or TSNGRQCAGIRP (SEQ ID NO: 134). In yet another aspect of thisembodiment, the N-terminal peptidyl fragment comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 126, SEQ IDNO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131,and SEQ ID NO: 132.

In yet another embodiment, the variable regions and conserved blocks ofa first Cry protein active against coleopteran insects are used to makethe eHIP of the invention in combination with variable regions andconserved blocks of a second Cry protein active against a lepidopteraninsect. Coleopteran active Cry proteins include but are not limited toCry3, Cry7, Cry8, and Cry34/Cry35. The lepidopteran active Cry proteinsinclude but are not limited to Cry1 and Cry9. In one aspect of thisembodiment, the first Cry protein is a Cry3A and the second Cry proteinis a Cry1A. In another aspect, the Cry3A protein can be replaced with amodified Cry3A, for example without limitation, the Cry3A055 proteindisclosed in U.S. Pat. No. 5,659,123, which is herein incorporated byreference. In still another aspect of this embodiment, the Cry3A proteinis a Cry3Aa and the Cry1A protein is a Cry1Aa or a Cry1Ab. In anotheraspect of this embodiment, the Cry3Aa is selected from the followinggroup and has the indicated GenBank Accession Number: Cry3Aa1 (M22472),Cry3Aa2 (J02978), Cry3Aa3 (Y00420), Cry3Aa4 (M30503), Cry3Aa5 (M37207),Cry3Aa6 (U10985), Cry3Aa7 (AJ237900), Cry3Aa8 (AAS79487), Cry3Aa9(AAW05659), Cry3Aa10 (AAU29411), and Cry3Aa11 (AY882576). In anotheraspect of this embodiment the Cry1Aa is selected from the followinggroup and has the indicated GenBank Accession Number: Cry1Aa1 (M11250),Cry1Aa2 (M10917), Cry1Aa3 (D00348), Cry1Aa4 (X13535), Cry1Aa5 (D17518),Cry1Aa6 (U43605), Cry1Aa7 (AF081790), Cry1Aa8 (I26149), Cry1Aa9(AB026261), Cry1Aa10 (AF154676), Cry1Aa11 (Y09663), Cry1Aa12 (AF384211),Cry1Aa13 (AF510713), Cry1Aa14 (AY197341), and Cry1Aa15 (DQ062690). Instill another aspect of this embodiment, the Cry1Ab is selected from thefollowing group and has the indicated GenBank Accession Number: Cry1Ab1(M13898), Cry1Ab2 (M12661), Cry1Ab3 (M15271), Cry1Ab4 (D00117), Cry1Ab5(X04698), Cry1Ab6 (M37263), Cry1Ab7 (X13233), Cry1Ab8 (M16463), Cry1Ab9(X54939), Cry1Ab10 (A29125), Cry1Ab11 (112419), Cry1Ab12 (AF059670),Cry1Ab13 (AF254640), Cry1Ab14 (U94191), Cry1Ab15 (AF358861), Cry1Ab16(AF37560), Cry1Ab17 (AAT46415), Cry1Ab18 (AAQ88259), Cry1Ab19(AY847289), Cry1Ab20 (DQ241675), Cry1Ab21 (EF683163), and Cry1Ab22(ABW87320). In yet another aspect of this embodiment, the first Cryprotein comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 68, SEQ ID NO: 70, and SEQ ID NO: 135, and thesecond Cry protein comprises an amino acid sequence set forth in SEQ IDNO: 72.

In one embodiment, the present invention encompasses an eHIP of theinvention comprising at least one crossover position between theN-terminal region of the first Cry protein and the C-terminal region ofthe second Cry protein located in conserved block 3, variable region 4,or conserved block 4. In one aspect of this embodiment, the crossoverposition in conserved block 3 is located immediately following an aminoacid corresponding to Ser451, Phe454, or Leu468 of SEQ ID NO: 70. Inanother aspect of this embodiment, the crossover position is located inconserved block 3 immediately following Ser451, Phe454, or Leu468 of SEQID: 70 or Ser450, Phe453, or Leu467 of SEQ ID NO: 68; or Ser497, Phe100,Leu114 of SEQ ID NO: 135. The crossover positions in certainCry3A/Cry1Ab eHIP embodiments or modified Cry3A/Cry1Ab eHIP embodimentsaccording to the invention are indicated on FIG. 2, which indicatespercent identity.

In another embodiment, an eHIP of the invention comprises at least twocrossover positions between an amino acid sequence from a first Bt Cryprotein and an amino acid sequence from the second Bt Cry protein. Inone aspect of this embodiment, a crossover position between a Cry3A ormodified Cry3A and a Cry1Ab or a Cry1Aa is located in conserved block 2immediately following an amino acid corresponding to Asp232 of SEQ IDNO: 70 and a second crossover position between Cry1Ab and Cry3A ormodified Cry3A is located in conserved block 3 immediately following anamino acid corresponding to Leu476 of SEQ ID NO: 72. In another aspectof this embodiment, a crossover position between a Cry3A or modifiedCry3A and a Cry1Ab or a Cry1Aa is located in conserved block 2immediately following Asp232 of SEQ ID NO: 70, or Asp231 of SEQ ID NO:68, or Asp278 of SEQ ID NO: 135, and a second crossover position betweenCry1Ab and Cry3A or modified Cry3A is located in conserved block 3immediately following Leu476 of SEQ ID NO: 72.

In still another aspect of this embodiment, a first crossover positionbetween a Cry3A or modified Cry3A and a Cry1Ab is located in conservedblock 3 immediately following an amino acid corresponding to Leu468 ofSEQ ID NO: 70 and a second crossover position between a Cry1Ab and aCry3A or modified Cry3A is located in conserved block 4 immediatelyfollowing an amino acid corresponding to Ile527 of SEQ ID NO: 72. Inanother aspect of this embodiment, the first crossover position betweena Cry3A or modified Cry3Aa and a Cry1Ab is located in conserved block 3immediately following an Leu468 of SEQ ID NO: 70, or Leu467 of SEQ IDNO: 68, or Leu114 of SEQ ID NO: 135, and the second crossover positionbetween a Cry1Ab and a Cry3A or modified Cry3A is located in conservedblock 4 immediately following Ile527 of SEQ ID NO: 72. In yet anotheraspect of this embodiment, the eHIP comprises the amino acid sequence ofSEQ ID NO: 28 or SEQ ID NO: 34.

In one embodiment, the present invention encompasses an eHIP wherein thefirst Cry protein is a Cry3A or a modified Cry3A and the second Cryprotein is a Cry1Aa or Cry1Ab and wherein the eHIP comprises an aminoacid sequence that has at least 80% identity to SEQ OD NO: 64. Inanother embodiment the eHIP comprises an amino acid sequence that has atleast 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98 or 99% identity to SEQ ID NO: 64. An alignment of certain eHIPembodiments of the invention with SEQ ID NO: 64 is shown in FIG. 2,which indicates percent identity.

In another embodiment, the present invention encompasses an eHIP whereinthe first Cry protein is a Cry3A or a modified Cry3A and the second Cryprotein is a Cry1Aa or Cry1Ab and wherein the eHIP comprises an aminoacid sequence that has at least 75% identity to SEQ OD NO: 70. Inanother embodiment the eHIP comprises an amino acid sequence that has atleast 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO: 70. Analignment of certain eHIP embodiments of the invention with SEQ ID NO:70 is shown in FIG. 1, which indicates percent identity.

In another embodiment, the present invention encompasses an eHIP havinga first crossover position between Cry3A or modified Cry3A and Cry1Aa orCry1Ab in conserved block 2 and a second crossover position betweenCry1Aa or Cry1Ab and Cry3A or modified Cry3A in conserved block 3 andwherein the eHIP comprises an amino acid sequence that has at least 56%identity to SEQ OD NO: 64. In one aspect of this embodiment, the eHIPhas at least 60, 70 or 80% identity to SEQ ID NO: 64. In another aspectof this embodiment, the eHIP has at least 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identity to SEQ ID NO:64.

In yet another embodiment, the present invention encompasses an eHIPcomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO:24, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 62; SEQ ID NO: 64, SEQ IDNO: 147, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 159 and SEQ ID NO:160.

In one embodiment, the eHIPs of the invention have activity againstother insect pests including but not limited to northern corn rootworm,Mexican corn rootworm, Colorado potato beetle, and/or European cornborer.

In another embodiment, the present invention encompasses a nucleic acidmolecule comprising a nucleotide sequence that encodes an eHIP of theinvention. In one aspect of this embodiment, the nucleic acid moleculecomprises a nucleotide sequence selected from the group consisting ofSEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO:23, SEQ ID NO: 29, SEQ ID NO: 33, SEQ ID NO: 61; SEQ ID NO: 63, SEQ IDNO: 146, SEQ ID NO: 152, SEQ ID NO: 154 and SEQ ID NO: 158. Specificallyexemplified teachings of methods to make nucleic acid molecules thatencode eHIPs can be found in Examples 1-41. Those skilled in the artwill recognize that modifications can be made to the exemplified methodsto make eHIPs encompassed by the present invention.

The present invention further encompasses expression cassettescomprising the nucleic acid molecules, and recombinant vectors andtransgenic non-human host cells, such as bacterial cells or plant cells,comprising the expression cassettes of the invention.

The present invention also encompasses recombinant vectors comprisingthe nucleic acids of this invention. In such vectors, the nucleic acidsare preferably comprised in expression cassettes comprising regulatoryelements for expression of the nucleotide sequences in a host cellcapable of expressing the nucleotide sequences. Such regulatory elementsusually comprise promoter and termination signals and preferably alsocomprise elements allowing efficient translation of polypeptides encodedby the nucleic acids of the present invention. Vectors comprising thenucleic acids are may be capable of replication in particular hostcells, preferably as extrachromosomal molecules, and are therefore usedto amplify the nucleic acids of this invention in the host cells. In oneembodiment, host cells for such vectors are microorganisms, such asbacteria, in particular Bacillus thuringiensis or E. coli. In anotherembodiment, host cells for such recombinant vectors are endophytes orepiphytes. In yet another embodiment, such vectors are viral vectors andare used for replication of the nucleotide sequences in particular hostcells, e.g. insect cells or plant cells. Recombinant vectors are alsoused for transformation of the nucleotide sequences of this inventioninto host cells, whereby the nucleotide sequences are stably integratedinto the DNA of a transgenic host. In one embodiment, the transgenichost is plant such as corn plant.

The eHIPs of the present invention have insect control activity whentested against insect pests in bioassays. In one embodiment, the eHIPsof the invention are active against coleopteran insects or lepidopteraninsects or both. In one aspect of this embodiment, the eHIPs of theinvention are active against western corn rootworm, northern cornrootworm, Mexican corn rootworm and/or Colorado potato beetle. Inanother aspect of this embodiment, the eHIPs of the invention are activeagainst European corn borer. The insect controlling properties of theeHIPs of the invention are further illustrated in Examples 43, 45 and46.

The present invention also encompasses a composition comprising aneffective insect-controlling amount of an eHIP according to theinvention.

In another embodiment, the invention encompasses a method of producing aeHIP that is active against insects, comprising: (a) obtaining a hostcell comprising a gene, which itself comprises a heterologous promotersequence operatively linked to a nucleic acid molecule of the invention;and (b) growing the transgenic host cell in such a manner to express aneHIP that is active against insects.

In yet another embodiment, the invention encompasses a method ofproducing an insect-resistant transgenic plant, comprising introducing anucleic acid molecule of the invention into the transgenic plant,wherein the nucleic acid molecule causes the expression of an eHIP inthe transgenic plant in an effective amount to control insects. In oneaspect of this embodiment, the insects are coleopteran insects orlepidopteran insects. In another aspect of this embodiment, thecoleopteran insects are western corn rootworm, northern corn rootworm,Mexican corn rootworm and/or Colorado potato beetle. In still anotheraspect of this embodiment, the lepidopteran insects are European cornborer.

In yet a further embodiment, the invention encompasses a method ofcontrolling insects, comprising delivering to the insects an effectiveamount of an eHIP of the invention. In one aspect of this embodiment,the insects are coleopteran insects or lepidopteran insects. In anotheraspect of this embodiment, the coleopteran insects are western cornrootworm, northern corn rootworm, Mexican corn rootworm and/or Coloradopotato beetle. In still another aspect of this embodiment, thelepidopteran insects are European corn borer. Typically, the eHIP isdelivered to the insects orally. In one aspect, the eHIP is deliveredorally through a transgenic plant comprising a nucleic acid sequencethat expresses an eHIP of the present invention.

The present invention further encompasses a method of controllinginsects wherein the transgenic plant further comprises a second nucleicacid molecule or groups of nucleic acid molecules that encode a secondpesticidal principle. Examples of such second nucleic acids are thosethat encode a Bt Cry protein, those that encode a VegetativeInsecticidal Protein, disclosed in U.S. Pat. Nos. 5,849,870 and5,877,012, incorporated herein by reference, or those that encode apathway for the production of a non-proteinaceous principle.

The present invention also encompasses a method of making an engineeredhybrid insecticidal protein (eHIP), comprising: (a) obtaining a first BtCry protein or modified Bt Cry protein; (b) obtaining a second Bt Cryprotein which is different from the first Bt Cry protein or modified BtCry protein; (c) combining complete or partial variable regions andconserved blocks of the first Bt Cry protein or modified Bt Cry proteinwith complete or partial variable regions and conserved blocks of thesecond Bt Cry protein to make an eHIP that has activity against at leastwestern corn rootworm; and optionally (d) inserting a peptidyl fragmentat the N-terminus or a protoxin tail region of a Bt Cry protein at theC-terminus of the eHIP, or both, wherein the N-terminal peptidylfragment or the C-terminal protoxin region or both confers activity uponthe eHIP, or increases the insecticidal activity of the eHIP or makesthe eHIP more stable than an eHIP without the peptidyl fragment orprotoxin tail region or both.

In another embodiment, the present invention encompasses a method ofmaking an engineered hybrid insecticidal protein (eHIP) comprising: (a)obtaining a first nucleic acid encoding a first Bt Cry protein ormodified Bt Cry protein and a second nucleic acid encoding a second Cryprotein different from the first Cry protein or modified Bt Cry protein;(b) isolating from the first and second nucleic acids, a nucleotidesequence that encodes complete or partial variable regions and conservedblocks of the first Bt Cry protein or modified Bt Cry protein and thesecond Bt Cry protein; (c) connecting together the resulting isolatednucleic acids of step (b) in such a way as to make a new hybrid nucleicacid that encodes a protein, and optionally fusing to a 5′ end of saidhybrid nucleic acid a nucleic acid that encodes a peptidyl fragmentresulting in a 5′ extension or fusing to a 3′ end of said hybrid nucleicacid a nucleic acid that encodes a protoxin tail region of a Bt Cryprotein resulting in a 3′ extension, or both; (d) inserting the hybridnucleic acid with or without one or both of the 5′ or 3′ extensions intoan expression cassette; (e) transforming the expression cassette into ahost cell, resulting in said host cell producing an eHIP; and (f)bioassaying the eHIP against at least western corn rootworm, whichresults in insecticidal activity against western corn rootworm.

In further embodiments of the methods of the invention, the first Bt Cryprotein or modified Bt Cry protein is a Cry3A or modified Cry3A and thesecond Bt Cry protein is A Cry1Aa or Cry1Ab.

In another embodiment of the methods of the invention, the N-terminalpeptidyl fragment comprises at 9 amino acids. In one aspect of thisembodiment the N-terminal peptidyl fragment comprises the amino acidsequence YDGRQQHRG (SEQ ID NO: 132) or the amino acid sequenceTSNGRQCAGIRP (SEQ ID NO: 133). In another aspect of this embodiment theN-terminal peptidyl fragment is selected from the group consisting ofSEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ IDNO: 130, SEQ ID NO: 131, and SEQ ID NO: 132.

In still another embodiment of the methods of the invention, theprotoxin tail region is from a Cry1Aa or Cry1Ab. In one aspect of thisembodiment, the protoxin tail region comprises at least 38 amino acids.In another aspect of this embodiment, the protoxin tail region comprisesan amino acid sequence that corresponds to amino acids 611-648 of SEQ IDNO: 72. In yet another aspect of this embodiment, the protoxin tailregion comprises amino acids 611-648 of SEQ ID NO: 72.

Specifically exemplified teachings of the methods of making the hybridnucleic acids and the eHIPs of the invention can be found in Examples1-41.

In further embodiments, the nucleotide sequences of the invention,particularly a sequence encoding the peptidyl fragment, the protoxintail, and/or conserved blocks 2, 3, and 4, can be further modified byincorporation of random mutations in a technique known as in vitrorecombination or DNA shuffling. This technique is described in Stemmeret al., Nature 370:389-391 (1994) and U.S. Pat. No. 5,605,793, which areincorporated herein by reference. Millions of mutant copies of anucleotide sequence are produced based on an original nucleotidesequence of this invention and variants with improved properties, suchas increased insecticidal activity, enhanced stability, or differentspecificity or ranges of target-insect pests are recovered. The methodencompasses forming a mutagenized double-stranded polynucleotide from atemplate double-stranded polynucleotide comprising a nucleotide sequenceof this invention, wherein the template double-stranded polynucleotidehas been cleaved into double-stranded-random fragments of a desiredsize, and comprises the steps of adding to the resultant population ofdouble-stranded random fragments one or more single or double-strandedoligonucleotides, wherein said oligonucleotides comprise an area ofidentity and an area of heterology to the double-stranded templatepolynucleotide; denaturing the resultant mixture of double-strandedrandom fragments and oligonucleotides into single-stranded fragments;incubating the resultant population of single-stranded fragments with apolymerase under conditions which result in the annealing of saidsingle-stranded fragments at said areas of identity to form pairs ofannealed fragments, said areas of identity being sufficient for onemember of a pair to prime replication of the other, thereby forming amutagenized double-stranded polynucleotide; and repeating the second andthird steps for at least two further cycles, wherein the resultantmixture in the second step of a further cycle includes the mutagenizeddouble-stranded polynucleotide from the third step of the previouscycle, and the further cycle forms a further mutagenized double-strandedpolynucleotide. In a preferred embodiment, the concentration of a singlespecies of double-stranded random fragment in the population ofdouble-stranded random fragments is less than 1% by weight of the totalDNA. In a further preferred embodiment, the template double-strandedpolynucleotide comprises at least about 100 species of polynucleotides.In another preferred embodiment, the size of the double-stranded randomfragments is from about 5 bp to 5 kb. In a further preferred embodiment,the fourth step of the method comprises repeating the second and thethird steps for at least 10 cycles.

As biological insect control agents, the eHIPs are produced byexpression of the nucleic acids in heterologous host cells capable ofexpressing the nucleic acids. In one embodiment, B. thuringiensis cellscomprising modifications of a nucleic acid of this invention are made.Such modifications encompass mutations or deletions of existingregulatory elements, thus leading to altered expression of the nucleicacid, or the incorporation of new regulatory elements controlling theexpression of the nucleic acid. In another embodiment, additional copiesof one or more of the nucleic acids are added to Bacillus thuringiensiscells either by insertion into the chromosome or by introduction ofextrachromosomally replicating molecules containing the nucleic acids.

In another embodiment, at least one of the nucleic acids of theinvention is inserted into an appropriate expression cassette,comprising a promoter and termination signal. Expression of the nucleicacid is constitutive, or an inducible promoter responding to varioustypes of stimuli to initiate transcription is used. In anotherembodiment, the cell in which the eHIP is expressed is a microorganism,such as a virus, bacteria, or a fungus. In yet another embodiment, avirus, such as a baculovirus, contains a nucleic acid of the inventionin its genome and expresses large amounts of the correspondinginsecticidal protein after infection of appropriate eukaryotic cellsthat are suitable for virus replication and expression of the nucleicacid. The insecticidal protein thus produced is used as an insecticidalagent. Alternatively, baculoviruses engineered to include the nucleicacid are used to infect insects in vivo and kill them either byexpression of the insecticidal toxin or by a combination of viralinfection and expression of the insecticidal toxin.

Bacterial cells are also hosts for the expression of the nucleic acidsof the invention. In one embodiment, non-pathogenic symbiotic bacteria,which are able to live and replicate within plant tissues, so-calledendophytes, or non-pathogenic symbiotic bacteria, which are capable ofcolonizing the phyllosphere or the rhizosphere, so-called epiphytes, areused. Such bacteria include bacteria of the genera Agrobacterium,Alcaligenes, Azospirillum, Azotobacter, Bacillus, Clavibacter,Enterobacter, Envinia, Flavobacter, Klebsiella, Pseudomonas, Rhizobium,Serratia, Streptomyces and Xanthomonas. Symbiotic fungi, such asTrichoderma and Gliocladium are also possible hosts for expression ofthe inventive nucleic acids for the same purpose.

Techniques for these genetic manipulations are specific for thedifferent available hosts and are known in the art. For example, theexpression vectors pKK223-3 and pKK223-2 can be used to expressheterologous genes in E. coli, either in transcriptional ortranslational fusion, behind the tac or trc promoter. For the expressionof operons encoding multiple ORFs, the simplest procedure is to insertthe operon into a vector such as pKK223-3 in transcriptional fusion,allowing the cognate ribosome binding site of the heterologous genes tobe used. Techniques for overexpression in gram-positive species such asBacillus are also known in the art and can be used in the context ofthis invention (Quax et al. In:Industrial Microorganisms:Basic andApplied Molecular Genetics, Eds. Baltz et al., American Society forMicrobiology, Washington (1993)). Alternate systems for overexpressionrely for example, on yeast vectors and include the use of Pichia,Saccharomyces and Kluyveromyces (Sreekrishna, In:Industrialmicroorganisms:basic and applied molecular genetics, Baltz, Hegeman, andSkatrud eds., American Society for Microbiology, Washington (1993);Dequin & Barre, Biotechnology L2:173-177 (1994); van den Berg et al.,Biotechnology 8:135-139 (1990)).

In one embodiment, at least one of the eHIPs of the invention isexpressed in a higher organism such as a plant. In this case, transgenicplants expressing effective amounts of the eHIP protect themselves frominsect pests. When the insect starts feeding on such a transgenic plant,it also ingests the expressed eHIP. This will deter the insect fromfurther biting into the plant tissue or may even harm or kill theinsect. A nucleic acid of the present invention is inserted into anexpression cassette, which may then be stably integrated in the genomeof the plant. In another embodiment, the nucleic acid is included in anon-pathogenic self-replicating virus. Plants transformed in accordancewith the present invention may be monocots or dicots and include, butare not limited to, corn, wheat, barley, rye, sweet potato, bean, pea,chicory, lettuce, cabbage, cauliflower, broccoli, turnip, radish,spinach, asparagus, onion, garlic, pepper, celery, squash, pumpkin,hemp, zucchini, apple, pear, quince, melon, plum, cherry, peach,nectarine, apricot, strawberry, grape, raspberry, blackberry, pineapple,avocado, papaya, mango, banana, soybean, tomato, sorghum, sugarcane,sugar beet, sunflower, rapeseed, clover, tobacco, carrot, cotton,alfalfa, rice, potato, eggplant, cucumber, Arabidopsis, and woody plantssuch as coniferous and deciduous trees.

Once a desired nucleic acid has been transformed into a particular plantspecies, it may be propagated in that species or moved into othervarieties of the same species, particularly including commercialvarieties, using traditional breeding techniques.

A nucleic acid of this invention is preferably expressed in transgenicplants, thus causing the biosynthesis of the corresponding eHIP in thetransgenic plants. In this way, transgenic plants with enhancedresistance to insects, particularly corn rootworm, are generated. Fortheir expression in transgenic plants, the nucleic acids of theinvention may require other modifications and optimization. Although inmany cases genes from microbial organisms can be expressed in plants athigh levels without modification, low expression in transgenic plantsmay result from microbial nucleic acids having codons that are notpreferred in plants. It is known in the art that all organisms havespecific preferences for codon usage, and the codons of the nucleicacids described in this invention can be changed to conform with plantpreferences, while maintaining the amino acids encoded thereby.Furthermore, high expression in plants is best achieved from codingsequences that have at least about 35% GC content, preferably more thanabout 45%, more preferably more than about 50%, and most preferably morethan about 60%. Microbial nucleic acids that have low GC contents mayexpress poorly in plants due to the existence of ATTTA motifs that maydestabilize messages, and AATAAA motifs that may cause inappropriatepolyadenylation. Although preferred gene sequences may be adequatelyexpressed in both monocotyledonous and dicotyledonous plant species,sequences can be modified to account for the specific codon preferencesand GC content preferences of monocotyledons or dicotyledons as thesepreferences have been shown to differ (Murray et al. Nucl. Acids Res.17:477-498 (1989)). In addition, the nucleic acids are screened for theexistence of illegitimate splice sites that may cause messagetruncation. All changes required to be made within the nucleic acidssuch as those described above are made using well known techniques ofsite directed mutagenesis, PCR, and synthetic gene construction usingthe methods described in the published patent applications EP 0 385 962,EP 0 359 472, and WO 93/07278.

In one embodiment of the invention an eHIP coding sequence and/or aparent Bt Cry protein coding sequence is/are made according to theprocedure disclosed in U.S. Pat. No. 5,625,136, herein incorporated byreference. In this procedure, maize preferred codons, i.e., the singlecodon that most frequently encodes that amino acid in maize, are used.The maize preferred codon for a particular amino acid might be derived,for example, from known gene sequences from maize. Maize codon usage for28 genes from maize plants is found in Murray et al., Nucleic AcidsResearch 17:477-498 (1989), the disclosure of which is incorporatedherein by reference.

In this manner, the nucleotide sequences can be optimized for expressionin any plant. It is recognized that all or any part of the gene sequencemay be optimized or synthetic. That is, synthetic or partially optimizedsequences may also be used.

For efficient initiation of translation, sequences adjacent to theinitiating methionine may require modification. For example, they can bemodified by the inclusion of sequences known to be effective in plants.Joshi has suggested an appropriate consensus for plants (NAR15:6643-6653 (1987)) and Clonetech suggests a further consensustranslation initiator (1993/1994 catalog, page 210). These consensusesare suitable for use with the nucleic acids of this invention. Thesequences are incorporated into constructions comprising the nucleicacids, up to and including the ATG (whilst leaving the second amino acidunmodified), or alternatively up to and including the GTC subsequent tothe ATG (with the possibility of modifying the second amino acid of thetransgene).

Expression of the nucleic acids in transgenic plants is driven bypromoters that function in plants. The choice of promoter will varydepending on the temporal and spatial requirements for expression, andalso depending on the target species. Thus, expression of the nucleicacids of this invention in leaves, in stalks or stems, in ears, ininflorescences (e.g. spikes, panicles, cobs, etc.), in roots, and/orseedlings is preferred. In many cases, however, protection against morethan one type of insect pest is sought, and thus expression in multipletissues is desirable. Although many promoters from dicotyledons havebeen shown to be operational in monocotyledons and vice versa, ideallydicotyledonous promoters are selected for expression in dicotyledons,and monocotyledonous promoters for expression in monocotyledons.However, there is no restriction to the provenance of selectedpromoters; it is sufficient that they are operational in driving theexpression of the nucleic acids in the desired cell.

In one embodiment promoters are used that are expressed constitutivelyincluding the actin or ubiquitin or cmp promoters or the CaMV 35S and19S promoters. The nucleic acids of this invention can also be expressedunder the regulation of promoters that are chemically regulated. Thisenables the eHIPs to be synthesized only when the crop plants aretreated with the inducing chemicals. Preferred technology for chemicalinduction of gene expression is detailed in the published application EP0 332 104 (to Ciba-Geigy) and U.S. Pat. No. 5,614,395. A preferredpromoter for chemical induction is the tobacco PR-la promoter.

In another embodiment a category of promoters which is wound induciblecan be used. Numerous promoters have been described which are expressedat wound sites and also at the sites of phytopathogen infection.Ideally, such a promoter should only be active locally at the sites ofinfection, and in this way the eHIPs only accumulate in cells that needto synthesize the eHIPs to kill the invading insect pest. Preferredpromoters of this kind include those described by Stanford et al. Mol.Gen. Genet. 215:200-208 (1989), Xu et al. Plant Molec. Biol. 22:573-588(1993), Logemann et al. Plant Cell 1:151-158 (1989), Rohrmeier & Lehle,Plant Molec. Biol. 22:783-792 (1993), Firek et al. Plant Molec. Biol.22:129-142 (1993), and Warner et al. Plant J. 3:191-201 (1993).

Tissue-specific or tissue-preferential promoters useful for theexpression of genes encoding eHIPs in plants, particularly corn, arethose which direct expression in root, pith, leaf or pollen,particularly root. Such promoters, e.g. those isolated from PEPC ortrpA, are disclosed in U.S. Pat. No. 5,625,136, or MTL, disclosed inU.S. Pat. No. 5,466,785. Both U.S. patents are herein incorporated byreference in their entirety.

Further embodiments are transgenic plants expressing the nucleic acidsin a wound-inducible or pathogen infection-inducible manner.

In addition to promoters, a variety of transcriptional terminators arealso available for use in hybrid nucleic acid construction using theeHIP genes of the present invention. Transcriptional terminators areresponsible for the termination of transcription beyond the transgeneand its correct polyadenylation. Appropriate transcriptional terminatorsand those that are known to function in plants include the CaMV 35Sterminator, the tml terminator, the nopaline synthase (NOS) terminator,the pea rbcS E9 terminator and others known in the art. These can beused in both monocotyledons and dicotyledons. Any available terminatorknown to function in plants can be used in the context of thisinvention.

Numerous other sequences can be incorporated into expression cassettesdescribed in this invention. These include sequences that have beenshown to enhance expression such as intron sequences (e.g. from Adhl andbronzel) and viral leader sequences (e.g. from TMV, MCMV and AMV).

It may be preferable to target expression of the nucleic acids of thepresent invention to different cellular localizations in the plant. Insome cases, localization in the cytosol may be desirable, whereas inother cases, localization in some subcellular organelle may bepreferred. Subcellular localization of transgene-encoded enzymes isundertaken using techniques well known in the art. Typically, the DNAencoding the target peptide from a known organelle-targeted gene productis manipulated and fused upstream of the nucleic acid. Many such targetsequences are known for the chloroplast and their functioning inheterologous constructions has been shown. The expression of the nucleicacids of the present invention is also targeted to the endoplasmicreticulum or to the vacuoles of the host cells. Techniques to achievethis are well known in the art.

Vectors suitable for plant transformation are described elsewhere inthis specification. For Agrobacterium-mediated transformation, binaryvectors or vectors carrying at least one T-DNA border sequence aresuitable, whereas for direct gene transfer any vector is suitable andlinear DNA containing only the construction of interest may bepreferred. In the case of direct gene transfer, transformation with asingle DNA species or co-transformation can be used (Schocher et al.Biotechnology 4:1093-1096 (1986)). For both direct gene transfer andAgrobacterium-mediated transfer, transformation is usually (but notnecessarily) undertaken with a selectable marker that may provideresistance to an antibiotic (kanamycin, hygromycin or methotrexate) or aherbicide (basta). Plant transformation vectors comprising the eHIPgenes of the present invention may also comprise genes (e.g.phosphomannose isomerase; PMI) which provide for positive selection ofthe transgenic plants as disclosed in U.S. Pat. Nos. 5,767,378 and5,994,629, herein incorporated by reference. The choice of selectablemarker is not, however, critical to the invention.

In another embodiment, a nucleic acid of the present invention isdirectly transformed into the plastid genome. A major advantage ofplastid transformation is that plastids are generally capable ofexpressing bacterial genes without substantial codon optimization, andplastids are capable of expressing multiple open reading frames undercontrol of a single promoter. Plastid transformation technology isextensively described in U.S. Pat. Nos. 5,451,513, 5,545,817, and5,545,818, in PCT application no. WO 95/16783, and in McBride et al.(1994) Proc. Nati. Acad. Sci. USA 91, 7301-7305. The basic technique forchloroplast transformation involves introducing regions of clonedplastid DNA flanking a selectable marker together with the gene ofinterest into a suitable target tissue, e.g., using biolistics orprotoplast transformation (e.g., calcium chloride or PEG mediatedtransformation). The 1 to 1.5 kb flanking regions, termed targetingsequences, facilitate homologous recombination with the plastid genomeand thus allow the replacement or modification of specific regions ofthe plastome. Initially, point mutations in the chloroplast 16S rRNA andrps12 genes conferring resistance to spectinomycin and/or streptomycinare utilized as selectable markers for transformation (Svab, Z.,Hajdukiewicz, P., and Maliga, P. (1990) Proc. Nati. Acad. Sci. USA 87,8526-8530; Staub, J. M., and Maliga, P. (1992) Plant Cell 4, 39-45).This resulted in stable homoplasmic transformants at a frequency ofapproximately one per 100 bombardments of target leaves. The presence ofcloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub, J. M., andMaliga, P. (1993) EMBO J. 12, 601-606). Substantial increases intransformation frequency are obtained by replacement of the recessiverRNA or r-protein antibiotic resistance genes with a dominant selectablemarker, the bacterial aadA gene encoding the spectinomycin-detoxifyingenzyme aminoglycoside-3′-adenyltransf erase (Svab, Z., and Maliga, P.(1993) Proc. Natl. Acad. Sci. USA 90, 913-917). Previously, this markerhad been used successfully for high-frequency transformation of theplastid genome of the green alga Chlamydomonas reinhardtii(Goldschmidt-Clermont, M. (1991) Nucl. Acids Res. 19:4083-4089). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by homologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleic acid of the present invention is inserted into aplastid-targeting vector and transformed into the plastid genome of adesired plant host. Plants homoplastic for plastid genomes containing anucleic acid of the present invention are obtained, and arepreferentially capable of high expression of the nucleic acid.

The eHIPs of the invention can be used in combination with otherpesticidal principles (e.g. Bt Cry proteins) to increase pest targetrange. Furthermore, the use of the eHIPs of the invention in combinationwith modified Cry3A toxins, Bt Cry proteins, or other CRW-activeprinciples, such as an RNAi, which have a different mode of action ortarget a different receptor in the insect gut, has particular utilityfor the prevention and/or management of corn rootworm resistance. Otherinsecticidal principles include, but are not limited to, lectins,α-amylase, peroxidase, and cholesterol oxidase. Vip genes, as disclosedin U.S. Pat. No. 5,889,174 and herein incorporated by reference, arealso useful in combination with the eHIPs of the present invention.

This co-expression of more than one insecticidal principle in the sametransgenic plant can be achieved by making a single recombinant vectorcomprising coding sequences of more than one insecticidal principle in aso called molecular stack and genetically engineering a plant to containand express all the insecticidal principles in the transgenic plant.Such molecular stacks may be also be made by using mini-chromosomes asdescribed, for example in U.S. Pat. No. 7,235,716. Alternatively, atransgenic plant comprising one nucleic acid encoding a firstinsecticidal principle can be re-transformed with a different nucleicacid encoding a second insecticidal principle and so forth.Alternatively, a plant, Parent 1, can be genetically engineered for theexpression of genes of the present invention. A second plant, Parent 2,can be genetically engineered for the expression of a supplementalinsect control principle. By crossing Parent 1 with Parent 2, progenyplants are obtained which express all the genes introduced into Parents1 and 2.

Transgenic seed of the present invention can also be treated with aninsecticidal seed coating as described in U.S. Pat. Nos. 5,849,320 and5,876,739, herein incorporated by reference. Where both the insecticidalseed coating and the transgenic seed of the invention are active againstthe same target insect, the combination is useful (i) in a method forenhancing activity of a eHIP of the invention against the target insectand (ii) in a method for preventing development of resistance to a eHIPof the invention by providing a second mechanism of action against thetarget insect. Thus, the invention provides a method of enhancingactivity against or preventing development of resistance in a targetinsect, for example corn rootworm, comprising applying an insecticidalseed coating to a transgenic seed comprising one or more eHIPs of theinvention.

Even where the insecticidal seed coating is active against a differentinsect, the insecticidal seed coating is useful to expand the range ofinsect control, for example by adding an insecticidal seed coating thathas activity against lepidopteran insects to the transgenic seed of theinvention, which has activity against coleopteran insects, the coatedtransgenic seed produced controls both lepidopteran and coleopteraninsect pests.

EXAMPLES

The invention will be further described by reference to the followingdetailed examples. These examples are provided for the purposes ofillustration only, and are not intended to be limiting unless otherwisespecified. Standard recombinant DNA and molecular cloning techniquesused here are well known in the art and are described by J. Sambrook, etal., Molecular Cloning: A Laboratory Manual, 3d Ed., Cold Spring Harbor,N.Y.: Cold Spring Harbor Laboratory Press (2001); by T. J. Silhavy, M.L. Berman, and L. W. Enquist, Experiments with Gene Fusions, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, New York, John Wiley andSons Inc., (1988), Reiter, et al., Methods in Arabidopsis Research,World Scientific Press (1992), and Schultz et al., Plant MolecularBiology Manual, Kluwer Academic Publishers (1998).

Example 1. Parental Coding Sequences

Maize optimized cry3Aa, cry1Ab, cry1Ba, and cry1Fa coding sequences;designated herein mocry3Aa, mocry1Ab, mocry1Ba and mocry1Fa,respectively, were made according to the procedure disclosed in U.S.Pat. No. 5,625,136, herein incorporated by reference in its entirety.

The cry3A055 (SEQ ID NO: 67) coding sequence, which encodes a Cry3A055protein (SEQ ID NO: 68) was made by modifying the mocry3A codingsequence by inserting a nucleotide sequence that encodes a Cathepsin Gprotease recognition site into domain I according to U.S. Pat. No.7,030,295, herein incorporated by reference in its entirety.

The mocry3Aa (SEQ ID NO: 67), which encodes the protein set forth in SEQID NO: 68, cry3A055 (SEQ ID NO: 69), which encodes the protein set forthin SEQ ID NO: 70, mocry1Ab (SEQ ID NO: 71), which encodes the proteinset forth in SEQ ID NO: 72, mocry1Ba (SEQ ID NO: 73), which encodes theprotein set forth in SEQ ID NO: 74, mocry1Fa (SEQ ID NO: 75), whichencodes the protein set forth in SEQ ID NO: 76, cry8Aa (SEQ ID NO: 77),which encodes the protein set forth in SEQ ID NO: 78, cry1Ac (SEQ ID NO:79), which encodes the protein set forth in SEQ ID NO: 80, and cry1Ia(SEQ ID NO: 81), which encodes the protein set forth in SEQ ID NO: 82,were used in the construction of the hybrid nucleic acids and theproteins which they encode and described in the following Examples.

Example 2. Use of PCR Primers to Construct Hybrid Nucleic Acids

Polymerase Chain Reaction (PCR) is a repetitive, enzymatic, primedsynthesis of a nucleic acid sequence. This procedure is well known andcommonly used by those skilled in this art (See Mullis, U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159; Saiki, Randall K., Stephen Scharf,Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, NormanArnheim [1985] “Enzymatic Amplification of .beta.-Globin GenomicSequences and Restriction Site Analysis for Diagnosis of Sickle CellAnemia,” Science 230:1350-1354.). PCR is based on the enzymaticamplification of a DNA fragment of interest that is flanked by twooligonucleotide primers that hybridize to opposite strands of the targetsequence. The primers are oriented with the 3′ ends pointing towardseach other. Repeated cycles of heat denaturation of the template,annealing of the primers to their complementary sequences, and extensionof the annealed primers with a DNA polymerase result in theamplification of the segment defined by the 5′ ends of the PCR primers.Since the extension product of each primer can serve as a template forthe other primer, each cycle essentially doubles the amount of DNAfragment produced in the previous cycle. This results in the exponentialaccumulation of the specific target fragment, up to several million-foldin a few hours. By using a thermostable DNA polymerase such as Taqpolymerase, which is isolated from the thermophilic bacterium Thermusaquaticus, the amplification process can be completely automated.

The chimeric coding sequences described in the following examples wereconstructed using various combinations of the exemplified primers shownin Table 1. The PCR reaction mixes and PCR thermocycling protocols usedin the experiments are listed in Tables 2 and 3, respectively. In eachof the examples that follow, the PCR primers are referred to by name and“SEQ ID NO:” and the PCR reaction mixes and PCR thermocycling protocolsare referred to by their respective numbers. It will be recognized bythe skilled person that other PCR primers and PCR reaction conditionscan be used to construct the chimeric coding sequences of the inventionand by listing the exemplified primers and PCR conditions that were usedin the instant invention is not meant to be limiting in any way.

TABLE 1 Primers used to construct the coding sequences encoding eHIPs.Primer Name Sequence SEQ ID NO: 5′3A-1-bam5′-CCGGATCCATGACGGCCGACAACAACACCGAGGC-3′ SEQ ID NO: 83 C3-3A-65′-CAGGGGCAGCTGGGTGATCT-3′ SEQ ID NO: 84 C3-1Ab-35′-AGATCACCCAGATCCCCCTG-3′ SEQ ID NO: 85 1Ab-6-sac5′-CCGAGCTCAGCTCCTACACCTGATCGATGTGGTAGTCGG-3′ SEQ ID NO: 86 8A-atg-delRI5′-CCGGATCCACCATGACTAGTAACGGCCGCCAGTGTGCTGGTATTCGCCCTTATGAC-3′SEQ ID NO: 87 C2-3A-4 5′-GTCCAGCACGGTCAGGGTCA-3′ SEQ ID NO: 88 reverse5′-GCGTGCAGTCAAGTCAGATC-3′ SEQ ID NO: 89 FR8a-OL-15′-GGTGTTGTTGTCGGCCGTCATAGGGCGAATACCAGCAC-3′ SEQ ID NO: 90 FR8a-OL-25′-GCCGACAACAACACCGAGGCCCTGGACAGCAGCACCACC-3′ SEQ ID NO: 91 C1-3A-25′-CAGGTGGGTGTTGGCGGCCTGGGCGTA-3′ SEQ ID NO: 92 5′FR8a5′-GGATCCACCATGACTAGTAAC-3′ SEQ ID NO: 93 5′FR8a-12aa5′-CCGGATCCACCATGTATGACGGCCGACAACAACACC-3′ SEQ ID NO: 94 C2-3A-35′-TGACCCTGACCGTGCTGGAC-3′ SEQ ID NO: 95 3′1Ab-dm35′-GAGCTCCTAGGTCACCTCGGCGGGCAC-3′ SEQ ID NO: 96 5′FR-del65′-GGATCCACCATGTGTGCTGGTATTCGCCCTAT-3′ SEQ ID NO: 97 5′1Ab-bam5′-CCGGATCCATGGACAACAACCCCAACATCAAC-3′ SEQ ID NO: 98 C3-3A-85′-GATGTCGCCGCCGGTGAAGC-3′ SEQ ID NO: 99 C3-3A-75′-GCTTCACCGGCGGCGACATC-3′ SEQ ID NO: 100 1B-55′-CCGCCGCGACCTGACCCTGGGCGTGCTGGAC-3′ SEQ ID NO: 101 1B-105′-CCGAGCTCCTAGAACAGGGCGTTCAC-3′ SEQ ID NO: 102 3A-225′-GGCCTTCACCAGGGGCAGCTGGGTGAT-3′ SEQ ID NO: 103 1B-75′-ATCACCCAGATCCCCATGGTGAAGGCC-3′ SEQ ID NO: 104 C3-1Ab-25′-CAGGGGGATCTGGGTGATCT-3′ SEQ ID NO: 105 C3-3A-55′-AGATCACCCAGCTGCCCCTG-3′ SEQ ID NO: 106 3A-12-sac5′-CCGAGCTCAGCTCAGATCTAGTTCACGGGGATGAACTCGATCTT-3′ SEQ ID NO: 107C4-3A-10 5′-TGGTGCTGGCGTAGTGGATGCGG-3′ SEQ ID NO: 108 C4-3A-95′-CCGCATCCACTACGCCAGCACCA-3′ SEQ ID NO: 109 C1-1Ab-15′-TACGTGCAGGCCGCCAACCTGCACCTG-3′ SEQ ID NO: 110 5′8Aa-dm35′-AGATCACCCAGCTGCCCCTGGTAAAGGGAGACATGTTATATC-3′ SEQ ID NO: 1113′8Aa-dm3 5′-GAGCTCCTATGTCTCATCTACTGGGATGAA-3′ SEQ ID NO: 112 tant-OL-25′-GAGGGTGTGGGCCTTCACCAGGGGCAGCTGGGT-3′ SEQ ID NO: 113 tant-OL-15′-ACCCAGCTGCCCCTGGTGAAGGCCCACACCCTC-3′ SEQ ID NO: 114 tant-3′sac5′-GAGCTCTAGCTTAAGCAGTCCACGAGGTT-3′ SEQ ID NO: 115 1Ac-OL-25′-TAAAAAGAAAGTTTCCCTTCACCAGGGGCAGCTGGGT-3′ SEQ ID NO: 116 1Ac-OL-15′-ACCCAGCTGCCCCTGGTGAAGGGAAACTTTCTTTTTA-3′ SEQ ID NO: 117 1Ac-3′sac5′-GAGCTCCTATGTTGCAGTAACTGGAATAAA-3′ SEQ ID NO: 118 1Ia-OL-25′-AAGACAGATTGAAAGCTTTTACTCAGGGGCAGCTGGGT-3′ SEQ ID NO: 119 1Ia-OL-15′-ACCCAGCTGCCCCTGAGTAAAAGCTTTCAATCTGTCTT-3′ SEQ ID NO: 120 1Ia-3′sac5′-GAGCTCCTACATGTTACGCTCAATATGGAGT-3′ SEQ ID NO: 121 FR-1Ab-25′-GATGTTGTTGAACTCGGCGCTCTTGTGGGTCCA-3′ SEQ ID NO: 122 FR-1Ab-15′-TGGACCCACAAGAGCGCCGAGTTCAACAACATC-3′ SEQ ID NO: 123 FR-1Ab-45′-GGCTCGTGGGGATGATGTTGTTGAAGTCGACGCTCTTGTGG-3′ SEQ ID NO: 124 FR-1Ab-35′-CCACAAGAGCGTCGACTTCAACACATCATCCCCAGCAGCC-3′ SEQ ID NO: 125 CMS945′-GGCGCGCCACCATGGCTAGCATGACTGGTGG-3′ SEQ ID NO: 136 CMS955′-GCAGGAACAGGTGGGTGTTG-3′ SEQ ID NO: 137 CMS965′-CCTGAACACCATCTGGCCCA-3′ SEQ ID NO: 138 CMS975′-CTGGCTGCTGGGGATGATGTTGTTGAAGTCGACGCTCTT-3′ SEQ ID NO: 139 CMS985′-GAGCTCTTAGGTCACCTCGGC-3′ SEQ ID NO: 140 CMS995′-AAGAGCGTCGACTTCAACAACATCATCCCCAGCAGCCAG-3′ SEQ ID NO: 141 CMS1005′-GAAGTACCGCGCCCGCATCCGCTACGCCAGCACCACCAAC-3′ SEQ ID NO: 142 CMS1015′-GTTGGTGGTGCTGGCGTAGCGGATGCGGGCGCGGTACTTC-3′ SEQ ID NO: 143

TABLE 2 PCR reaction mixes. Mix 1 Mix 2 Mix 3 50-100 ng template DNA50-100 ng template DNA 50-100 ng template DNA 0.8 μM primer 1 0.8 μMprimer 1 0.8 μM primer 1 0.8 μM primer 2 0.8 μM primer 2 0.8 μM primer 21X Pfu buffer 1X Taq buffer 1X cDNA Advantage buffer 0.4 mM dNTPs 0.4 mMdNTPs 0.4 mM dNTPs 2% formamide 2% formamide x units cDNA Advantage 1.25units Pfu Polymerase (Stratagene) 2.5 units Taq Polymerase (Qiagen)Polymerase (Clontech) 2.5 units Taq Polymerase water to a total volumeof 50 μl water to a total volume of 50 μl (Qiagen) water to a totalvolume of 50 μl Mix 4 Mix 5 50-100 ng template DNA 50-100 ng templateDNA 0.4 μM primer 1 0.4 μM primer 1 0.4 μM primer 2 0.4 μM primer 2 1XPCR buffer (Invitrogen) 1X Pfu buffer (Stratagene) 0.4 mM dNTPs 0.2 mMdNTPs 2.5 units HotStart Taq Polymerase 1.25 units Pfu Turbo Polymerasewater to a total volume of 50 μl water to a total volume of 50 μl

TABLE 3 PCR thermocycling profiles. Thermocycle Profile 1 ThermocycleProfile 2 Thermocycle Profile 3 94° C.-5 minutes 94° C.-5 minutes 94°C.-5 minutes 20 cycles: 20 cycles: 20 cycles: 94° C.-30 seconds 94°C.-30 seconds 94° C.-30 seconds 65° C.-30 seconds 55° C.-30 seconds 55°C.-30 seconds 72° C.-30 seconds 72° C.-30 seconds 68° C.-30 seconds 72°C.-7 minutes 72° C.-7 minutes 68° C.-7 minutes hold at 4° C. hold at 4°C. hold at 4° C. Thermocycle Profile 4 Thermocycle Profile 5 ThermocycleProfile 6 94° C.-15 minutes 94° C.-5 minutes 94° C.-5 minutes 20 cycles:20 cycles: 20 cycles: 94° C.-30 seconds 94° C.-30 seconds 94° C.-30seconds 50-70° C.-30 seconds 55-75° C.-30 seconds 55-75° C.-30 seconds72° C.-30 seconds 72° C.-1 minute 72° C.-2 minutes 72° C.-7 minutes 72°C.-15 minutes 72° C.-15 minutes hold at 4° C. hold at 4° C. hold at 4°C.

Table 4 shows the relationship between the three domains of Cry3A155,Cry1Ab and Cry3A with their respective variable regions and conservedblocks. The amino acids comprised in the domains, conserved blocks andvariable regions are shown for each protein.

TABLE 4

Example 3. Construction of 2OL-8a

A first nucleic acid fragment encoding an N-terminal portion of aCry3A055 protein (SEQ ID NO: 70) was PCR amplified from a plasmidcomprising cry3A055 (SEQ ID NO: 69) using primers 5′3A-1-bam (SEQ ID NO:83) and C3-3A-6 (SEQ ID NO: 84) and PCR reaction Mix 1 and thermocycleProfile 1. This PCR reaction introduced a point mutation by deletingnucleotide 28 of SEQ ID NO: 69 (cry3A055), which caused a frame shift inthe cry3A055 reading frame, and deleted the BamHI site and Kozaksequence (Kozak, M., 1986. Cell 44:283-92) at the 5′ end of theresulting amplicon.

A second nucleic acid fragment encoding a C-terminal portion of a Cry1Abprotein (SEQ ID NO: 72) was PCR amplified from a plasmid comprisingmocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO: 85) and1Ab-6-Sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycle Profile1.

The first and second nucleic acids described above were connected byusing them as templates in an overlap PCR reaction (Horton et al., 1989.Gene 77: 61-68) with the primers 5′3A-1-bam (SEQ ID NO: 83) and1Ab-6-Sac (SEQ ID NO: 86) using PCR reaction Mix 2 and thermocycleProfile 1, except a 45-65° C. gradient was used for the annealingtemperature.

The resulting amplicon was ligated as a blunt ended fragment to apCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) cut with Smal to formplasmid p2OL8a/CR2.1. A BamHI-SacI fragment from p2OL8a/CR2.1 was thenligated to pET21a (EMD Biosciences, Inc., San Diego, Calif.), which wascut with BamHI-SacI, and transformed into E. coli. The BamHI-SacIfragment from p2OL8a/CR2.1 comprised 40 nucleotides derived from thepCR2.1-TOPO vector adjacent to the out of frame amplicon from the firstPCR reaction. Ligating this BamHI-SacI fragment to pET21a created anopen reading frame starting with the start codon (ATG) of a T7 tag andending with the SacI site of the inserted DNA. This open reading framewas designated 2OL-8a (SEQ ID NO: 1) and encodes the 2OL-8a chimericinsecticidal protein (SEQ ID NO: 2). Thus, the 2OL-8a chimericinsecticidal protein comprises, from N-terminus to C-terminus, apeptidyl fragment comprising the amino acid sequenceMASMTGGQQMGRGSTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 126), amino acids 10-468of a Cry3A055 protein (SEQ ID NO: 70), which comprises variable regions1, conserved block 1, variable region 2, conserved block 2, variableregion 3, and the N-terminal 24 amino acids of conserved block 3, andamino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprisesthe C-terminal 24 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5, and variableregion 6; and 38 amino acids of the Cry1Ab protoxin tail region.

The nucleotides that encode amino acids 1-14 of the peptidyl fragmentare derived from the T7-tag and the BamHI cleavage site of the pET21avector. The nucleotides that encode amino acids 15-26 of the peptidylfragment are derived from the pCR2.1-TOPO vector. And the nucleotidesthat encode amino acids 27-35 of the peptidyl fragment are derived fromcry3A055 which are out of frame with the remainder of the cry3A055coding sequence.

Example 4. Construction of FR8a

The FR8a coding sequence was constructed by placing a Kozak sequence(ACC) and a start codon (ATG) just downstream of an N-terminal BamHIsite in 2OL-8a (See Example 3). In addition, an EcoRI site in 2OL-8a wasdisrupted to aid in future vectoring of FR8a. All of these changes weremade using one PCR reaction with 2OL-8a as the template and the primers:8a-atg-delRJ (SEQ ID NO: 87) and C2-3A-4 (SEQ ID NO: 88) using PCRreaction Mix 2 and thermocycle Profile 2.

The resulting amplicon was ligated to a pCR2.1-TOPO vector (Invitrogen).A BamHI-PpuMI fragment from the cloned PCR product was then ligated to aPpuMI-NcoI fragment from 2OL8a/pCR2.1 (See Example 3) and a NcoI-BamHIfragment from 2OL8a/pCR2.1 to create FR8a (SEQ ID NO: 3) which encodesthe FR8a chimeric insecticidal protein (SEQ ID NO: 4). Thus, the FR8achimeric insecticidal protein comprises, from N-terminus to C-terminus,a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of aCry3A055 protein (SEQ ID NO: 70), which comprises variable regions 1,conserved block 1, variable region 2, conserved block 2, variable region3, and the N-terminal 24 amino acids of conserved block 3, and aminoacids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 24 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5, and variableregion 6; and 38 amino acids of the Cry1Ab protoxin tail region.

The FR8a eHIP was very active against western corn rootworm as shown inTable 5. Therefore, elimination of the T7 amino acid sequence from theN-terminal peptidyl fragment from the 2OL-8a eHIP did not have anegative impact on insecticidal activity.

Adding an additional 34 amino acids to the N-terminus of FR8a created aeHIP, designated FR8a+34 (SEQ ID NO: 160), with an N-terminal peptidylfragment of 56 amino acids (SEQ ID NO: 131). The 56 amino acidN-terminal prptidyl fragment had no negative effect on activity of FR8aagainst western corn rootworm (See Table 5).

Example 5. Construction of FRCG

In order to determine if a cathepsin G protease recognition site wasnecessary for the insecticidal activity of a hybrid protein comprisingan N-terminal fragment of Cry3A055, a construct was made whicheliminated the cathepsin G site from the FR8a hybrid protein (Example4). A first MluI-PpuMI nucleic acid fragment from a plasmid comprisingFR8a (SEQ ID NO: 3) and a second PpuMI/MluI nucleic acid fragment from aplasmid comprising mocry3Aa (SEQ ID NO: 67) were ligated using standardmolecular biology techniques to create FRCG (also designated FR8a-catg)(SEQ ID NO: 5) which encodes the FRCG hybrid protein (SEQ ID NO: 6).Thus, the FRCG chimeric insecticidal protein comprises, from N-terminusto C-terminus, a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 of a Cry3Aprotein (SEQ ID NO: 68), which comprises variable regions 1, conservedblock 1, variable region 2, conserved block 2, variable region 3, andthe N-terminal 24 amino acids of conserved block 3, and amino acids477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 24 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5, and variableregion 6; and 38 amino acids of the Cry1Ab protoxin tail region.

The FRCG protein was as active against western corn rootworm as the FR8aprotein (See Table 5) suggesting that a cathepsin G protease site is notrequired for insecticidal activity of a eHIP.

Example 6. Construction of FR8a-9F

A first approximately 323 bp nucleic acid fragment was PCR amplifiedfrom a plasmid comprising FR8a (SEQ ID NO: 3) using primers reverse (SEQID NO: 89) and FR8a-OL-1 (SEQ ID NO: 90) and PCR reaction Mix 2 andthermocycle Profile 2. A second approximately 470 bp nucleic acidfragment was PCR amplified from a plasmid comprising FR8a using primersFR8a-OL-2 (SEQ ID NO: 91) and C1-3A-2 (SEQ ID NO: 92) and PCR reactionMix 2 and thermocycle Profile 2. The two resulting amplicons wereconnected by using them as templates in an overlap PCR reaction withprimers 5′FR8a (SEQ ID NO: 93) and C1-3A-2 (SEQ ID NO: 92) using PCRreaction Mix 2 and thermocycle Profile 2 to amplify the 5′ end ofFR8a-9F. The overlap PCR product was cloned into a pCR2.1-TOPO vector(Invitrogen) designated 5′FR-9F/pCR2.1. A BamHI/PpuMI fragment of5′FR-9F/pCR2.1 was then ligated to a PpuMI/BamHI fragment of FR8a tocreate FR8a-9F (SEQ ID NO: 7) which encodes the FR8a-9F chimeric protein(SEQ ID NO: 8). Thus, the FR8a-9F chimeric insecticidal proteincomprises, from N-terminus to C-terminus, a peptidyl fragment comprisingthe amino acid sequence MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids1-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises variableregions 1, conserved block 1, variable region 2, conserved block 2,variable region 3, and the N-terminal 24 amino acids of conserved block3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72), whichcomprises the C-terminal 24 amino acids of conserved block 3, variableregion 4, conserved block 4, variable region 5, conserved block 5, andvariable region 6; and 38 amino acids of the Cry1Ab protoxin tailregion.

The FR8a-9F eHIP was slightly less active against western corn rootwormthan the FR8a eHIP (See Table 5) suggesting that the C-terminal 9 aminoacids of the peptidyl fragment of SEQ ID NO: 127 play a role inconferring full insecticidal activity to FR8a.

Example 7. Construction of FR-9F-catg

The FR-9F-catg coding sequence was created to place the out-of-framecry3A055 derived nucleotides of FR8a back in frame and to eliminate thecathepsin G protease recognition site. A BamHI/PpuMI fragment of5′FR-9F/pCR2.1 (See Example 6) was ligated with a PpuMI/BamHI fragmentof FRCG (See Example 5) to create the FR-9F-catg coding sequence (SEQ IDNO: 9) which encodes the FR-9F-catg chimeric protein (SEQ ID NO: 10).Thus, the FR-9F-catg chimeric protein comprises, from N-terminus toC-terminus, a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRP (SEQ ID NO: 129), amino acids 1-467 of a Cry3Aa protein(SEQ ID NO: 68), which comprises variable regions 1, conserved block 1,variable region 2, conserved block 2, variable region 3, and theN-terminal 24 amino acids of conserved block 3, and amino acids 477-648of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24amino acids of conserved block 3, variable region 4, conserved block 4,variable region 5, conserved block 5, and variable region 6; and 38amino acids of the Cry1Ab protoxin tail region.

The FR8a-9F-catg eHIP provided the same level of activity as FR8aagainst western corn rootworm (See Table 5) confirming that an eHIP canbe made from either a modified Cry3A or a native Cry3 sequence.

Example 8. Construction of FR8a-12aa

The nucleotides encoding amino acids 2-13 of the peptidyl fragmentcomprised in FR8a (SEQ ID NO: 4) were removed using PCR. A fragment wasPCR amplified from a plasmid comprising FR8a (SEQ ID NO: 3) usingprimers 5′FR8a-12aa (SEQ ID NO: 94) and C1-3A-2 (SEQ ID NO: 90) and PCRreaction Mix 1 and thermocycle Profile 1. The resulting amplicon wascloned into pCR2.1-TOPO (Invitrogen). A BamHI-PpuMI fragment from thepCR2.1-TOPO clone was then ligated with a PpuMI-BamHI fragment from aplasmid comprising FR8a to create FR8a-12aa (SEQ ID NO: 11) whichencodes the FR8a-12aa chimeric insecticidal protein (SEQ ID NO: 12).Thus, the FR8a-12aa chimeric insecticidal protein comprises, fromN-terminus to C-terminus, a peptidyl fragment comprising the amino acidsequence MYDGRQQHRG (SEQ ID NO: 128), amino acids 10-468 of a Cry3A055protein (SEQ ID NO: 70), which comprises variable regions 1, conservedblock 1, variable region 2, conserved block 2, variable region 3, andthe N-terminal 24 amino acids of conserved block 3, and amino acids477-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 24 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5, and variableregion 6; and 38 amino acids of the Cry1Ab protoxin tail region.

The FR8a-12aa eHIP provided the same level of activity as FR8a againstwestern corn rootworm (See Table 5) suggesting that the N-terminal 12amino acids of the peptidyl fragment of SEQ ID NO: 127 are not necessaryfor full insecticidal activity of FR8a.

Example 9. Construction of Wr-9mut

A nucleic acid fragment was PCR amplified from FR8a/pCR2.1 (Example 2)using primers 5′FR8a-12aa (SEQ ID NO: 94) and C1-3A-2 (SEQ ID NO: 92)and PCR reaction Mix 1 and thermocycle Profile 2. The resulting ampliconwas cloned into pCR2.1-TOPO (Invitrogen). A BamHI/PpuMI fragment wasthen ligated to a PpuMI/BamHI fragment of FR8a (SEQ ID NO: 3) to createWr-9mut (SEQ ID NO: 13) which encodes the WR-9mut protein (SEQ ID NO:14), which comprises, from N-terminus to C-terminus, a peptidyl fragmentcomprising the amino acid sequence MYDGRQQHRG (SEQ ID NO: 128), andamino acids 10-598 of a Cry3A055 protein (SEQ ID NO: 70). Thus theWR-9mut protein is Cry3A055 with an N-terminal peptidyl fragment of theinvention.

The WR-9mut protein was not active against western corn rootworm.Therefore, the addition of an N-terminal peptidyl fragment to anon-hybrid modified Cry3a protein destroyed insecticidal activity. Thissuggests that there may be some interaction between the Cry1AbC-terminal portion of FR8a and the N-terminal peptidyl fragment thatconfers full insecticidal activity to FR8a.

Example 10. Construction of FRD3

The 3′ end of this coding sequence was made by PCR amplifying a fragmentfrom a plasmid comprising FR8a (SEQ ID NO: 3) using primers C2-3A-3 (SEQID NO: 95) and 3′1Ab-dm3 (SEQ ID NO: 96) and PCR reaction Mix 2 andthermocycle Profile 2. The resulting amplicon was cloned intopCR2.1-TOPO (Invitrogen). A 364 bp ApaI/SacI fragment of the clonedamplicon, designated 3′FRD3/pCR2.1, was ligated with a SacI/ApaIfragment of FR8a to create FRD3 (SEQ ID NO: 15) which encodes the FRD3chimeric protein (SEQ ID NO: 16). The FRD3 chimeric protein comprises,from N-terminus to C-terminus, a peptidyl fragment comprising the aminoacid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids10-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprises completevariable region 1, conserved block 1, variable region 2, conserved block2, variable region 3, and the N-terminal 24 amino acids of conservedblock 3, and amino acids 477-610 of a Cry1Ab protein (SEQ ID NO: 72),which comprises the C-terminal 24 amino acids of conserved block 3,variable region 4, conserved block 4, variable region 5, conserved block5, and variable region 6. Thus, the FRD3 chimeric insecticidal proteinis a variant of an FR8a chimeric insecticidal protein with the 38 aminoacid region of the Cry1Ab protoxin tail deleted.

The FRD3 eHIP provided the same level of activity as FR8a againstwestern corn rootworm (See Table 5) suggesting that the 38 amino acidprotoxin tail region of FR8a is not necessary for full insecticidalactivity.

Example 11. Construction of FR-12-cg-dm3

A 3082 bp SacI/PpuMI fragment from a plasmid comprising FR8a-12 (SeeExample 8), a 721 bp PpuMI/MluI fragment of FRCG (See Example 5) and a923 bp MluI/SacI fragment of FRD3 (See Example 10) were combined tocreate the FR-12-cg-dm3 coding sequence (SEQ ID NO: 17) which encodesthe FR-12-cg-dm3 chimeric protein (SEQ ID NO: 18). The FR-12-cg-dm3chimeric protein comprises, from N-terminus to C-terminus, a peptidylfragment comprising the amino acid sequence MYDGRQQHRG (SEQ ID NO: 129),amino acids 10-467 of a Cry3Aa protein (SEQ ID NO: 70), which comprisescomplete variable region 1, conserved block 1, variable region 2,conserved block 2, variable region 3, and the N-terminal 24 amino acidsof conserved block 3, and amino acids 477-610 of a Cry1Ab protein (SEQID NO: 72), which comprises the C-terminal 24 amino acids of conservedblock 3, variable region 4, conserved block 4, variable region 5,conserved block 5, and variable region 6. Thus, the FR-12-cg-dm3chimeric protein is a variant of FR8a with 12 N-terminal amino acids ofthe peptidyl fragment, the cathepsin G protease recognition site, andthe 38 amino acid region of the Cry1Ab protoxin tail deleted.

The FR-12-cg-dm3 eHIP was not as active against western corn rootworm asFR8a (See Table 5) suggesting that some interaction between theC-terminal portion of FR8a and the N-terminal peptidyl fragment isrequired for full insecticidal activity.

Example 12. Construction of 9F-cg-del6

The 5′ end of this coding sequence was made by PCR amplifying a fragmentfrom a plasmid comprising FR-9F-catg (See Example 7) using primers5′FR-del6 (SEQ ID NO: 97) and C1-3A-2 (SEQ ID NO: 92) and PCR reactionMix 3 and thermocycle Profile 3. The resulting amplicon was cloned intopCR2.1-TOPO. A 215 bp BamHI/PpuMI fragment was then ligated with a 4668bp PpuMI/BamHI fragment of FR-9F-catg to create FR-9F-cg-del6 (SEQ IDNO: 19) which encodes the FR-9F-cg-del6 chimeric protein (SEQ ID NO:20). The FR-9F-cg-del6 chimeric protein comprises, from N-terminus toC-terminus, a peptidyl fragment comprising the amino acid sequenceMCAGIRP (SEQ ID NO: 130), amino acids 1-467 of a Cry3A protein (SEQ IDNO: 68), which comprises variable regions 1, conserved block 1, variableregion 2, conserved block 2, variable region 3, and the N-terminal 24amino acids of conserved block 3, and amino acids 477-648 of a Cry1Abprotein (SEQ ID NO: 72), which comprises the C-terminal 24 amino acidsof conserved block 3, variable region 4, conserved block 4, variableregion 5, conserved block 5, and variable region 6; and 38 amino acidsof the Cry1Ab protoxin tail region. Thus, the FR-9F-cg-del6 chimericprotein is a variant of FR8a-9F-catg with amino acids 2 to 7 of thepeptidyl fragment deleted.

The FR-9F-cg-del6 was not active against western corn rootwormsuggesting that the N-terminal peptidyl fragment needs at least 7 aminoacids of the C-terminal 9 amino acids of SEQ ID NO: 127 to be activeagainst western corn rootworm.

Example 13. Construction of FR-cg-dm3

A 3839 bp MluI/SacI fragment of FRCG (Example 5) and a 923 bp MluI/SacIfragment of FRD3 (Example 10) were ligated to create FR-cg-dm3 (SEQ IDNO: 21) which encodes the FR-cg-dm protein (SEQ ID NO: 22). TheFR-cg-dm3 chimeric insecticidal protein comprises, from N-terminus toC-terminus, a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 of a Cry3Aprotein (SEQ ID NO: 68), which comprises variable regions 1, conservedblock 1, variable region 2, conserved block 2, variable region 3, andthe N-terminal 24 amino acids of conserved block 3, and amino acids477-610 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 24 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5, and variableregion 6.

The FRD3 eHIP the same level of activity against western corn rootwormas FR8a (See Table 5) confirming that the cathepsin G site and theprotoxin tail region of FR8a were not required for full insecticidalactivity against western corn rootworm.

Example 14. Construction of 9F-cg-dm3

A MluI/SacI fragment from a plasmid comprising FR-9F-cg (See Example 7)was ligated with a 923 bp MluI/SacI fragment from a plasmid comprisingFRD3 (See Example 10) to create 9F-cg-dm3 (SEQ ID NO: 23) which encodesthe 9F-cg-dm3 chimeric protein (SEQ ID NO: 24). The 9F-cg-dm3 proteincomprises, from N-terminus to C-terminus, a peptidyl fragment comprisingthe amino acid sequence MTSNGRQCAGIRP (SEQ ID NO: 129), amino acids1-467 of a Cry3A protein (SEQ ID NO: 68), which comprises variableregions 1, conserved block 1, variable region 2, conserved block 2,variable region 3, and the N-terminal 24 amino acids of conserved block3, and amino acids 477-610 of a Cry1Ab protein (SEQ ID NO: 72), whichcomprises the C-terminal 24 amino acids of conserved block 3, variableregion 4, conserved block 4, variable region 5, conserved block 5, andvariable region 6.

The 9F-cg-dm3 eHIP provided the same level of activity against westerncorn rootworm (See Table 5) confirming that the C-terminal 9 amino acidsof the peptidyl fragment could confer activity when domain I of the eHIPwas comprised of either modified Cry3A (Cry3A055) variable regions andconserved blocks or Cry3A variable regions and conserved blocks.

Example 15. Construction of B8a

A nucleic acid fragment encoding an N-terminal portion of a Cry3A055protein (SEQ ID NO: 70), was PCR amplified from a plasmid comprisingcry3A055 (SEQ ID NO: 69) using primers 5′3A-1-bam (SEQ ID NO: 83) andC3-3A-8 (SEQ ID NO: 99) and PCR reaction Mix 1 and thermocyclingProfile 1. A nucleic acid fragment encoding a C-terminal portion of aCry1Ab protein (SEQ ID NO: 72), comprising variable regions 4-6, wasamplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) usingprimers C3-3A-7 (SEQ ID NO: 100) and 1Ab-6-sac (SEQ ID NO: 86) and PCRreaction Mix 1 and thermocycling Profile 1. The resulting amplicon wasdesignated 2OL-8b.

A nucleic acid fragment encoding an N-terminal portion of the Cry3A055protein (SEQ ID NO: 70), was PCR amplified from a plasmid comprisingcry3A055 (SEQ ID NO: 69) using primers 5′3A-1-bam (SEQ ID NO: 83) andC2-3A-4 (SEQ ID NO: 88) and PCR reaction Mix 1 and thermocycling Profile1.

A nucleic acid fragment encoding a C-terminal portion of a Cry1Baprotein (SEQ ID NO: 74) was PCR amplified from a plasmid comprisingmocry1Ba (SEQ ID NO: 73) using primers 1B-5 (SEQ ID NO: 101) and 1B-10(SEQ ID NO: 102) and PCR reaction Mix 1 and thermocycling Profile 1,except a 60° C. annealing temperature was used.

The two above-described PCR products were then used as the templates inan overlap PCR reaction with primers 5′3A-1-bam (SEQ ID NO: 83) and1B-10 (SEQ ID NO: 102) using PCR reaction Mix 1 and thermocyclingProfile 2. The resulting amplicon was designated B10.

Next, a nucleic acid fragment of cry3A055 (SEQ ID NO: 69) was PCRamplified using 2OL-8b (see above) as the template and primers5′3A-1-bam (SEQ ID NO: 83) and 3A-22 (SEQ ID NO: 103) with the followingPCR conditions: Mix 1, thermocycling profile: 94° C.—45 seconds, 50°C.-70° C. gradient—45 seconds, 72° C.-90 seconds for 30 cycles. Anothernucleic acid fragment was PCR amplified using B10 (see above) as thetemplate and primers 1B-7 (SEQ ID NO: 104) and 1B-10 (SEQ ID NO: 102)using PCR reaction Mix 1 and thermocycling Profile 2, except a 60° C.annealing temperature was used. The two resulting PCR products were thenused as templates in an overlap PCR reaction with primers 5′3A-1-bam(SEQ ID NO: 83) and 1B-10 (SEQ ID NO: 102) using the following PCRconditions: Mix 2, thermocycling profile: 94° C.—30 seconds, 40° C.-60°C. gradient—30 seconds, 72° C.-60 seconds for 30 cycles.

The resulting PCR product was ligated to a pCR2.1-TOPO vector(Invitrogen) and designated B10/pCR2.1. A BamHi-SacI fragment fromB8a/pCR2.1 was then ligated to pET21a (Novagen), which was cut withBamHi/SacI, to create the B8a coding sequence (SEQ ID NO: 25), whichencodes a B8a hybrid toxin (SEQ ID NO: 26). The B8a hybrid proteincomprises, from N-terminus to C-terminus, amino acids 1-468 of aCry3A055 protein (SEQ ID NO: 70), and amino acids 505-656 of a Cry1Baprotein (SEQ ID NO: 74).

Example 16. Construction of 5*B8a

A BamHI-XbaI fragment from a plasmid comprising 2OL-8a (See Example 3)and a XbaI-SacI fragment from a plasmid comprising B8a (See Example 15)were ligated to create 5*B8a (SEQ ID NO: 27), which encodes the 5*B8achimeric protein (SEQ ID NO: 28). The 5*B8a protein comprises, fromN-terminus to C-terminus, a peptidyl fragment comprising the amino acidsequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-467 ofa Cry3A055 protein (SEQ ID NO: 70) and amino acids 505-656 of a Cry1Baprotein (SEQ ID NO: 74). Thus, the 5*B8a chimeric protein is the B8ahybrid protein to which an N-terminal peptidyl fragment has been added.

Example 17. Construction of V3A

This gene was PCR amplified using 3 fragments together as templates: thefirst fragment was amplified from a plasmid comprising cry3A055 (SEQ IDNO: 69) using primers 5′3A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO:88) and PCR reaction Mix 1 and thermocycling Profile 1; the secondfragment was amplified from a plasmid comprising mocry1Ab (SEQ ID NO:71) using primers C2-3A-3 (SEQ ID NO: 95) and C3-1Ab-2 (SEQ ID NO: 105)and PCR reaction Mix 1 and thermocycling Profile 1; and the thirdfragment was amplified from a plasmid comprising cry3A055 (SEQ ID NO:69) using primers C3-3A-5 (SEQ ID NO: 106) and 3A-12-sac (SEQ ID NO:107) and PCR reaction Mix 1 and thermocycling Profile 1. These 3 PCRproducts were then used as templates in an overlap PCR reaction withprimers 5′3A-1-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ ID NO: 107) usingPCR reaction Mix 1 and thermocycling Profile 1, to produce the v3Acoding sequence (SEQ ID NO: 29), which encodes the V3A hybrid protein(SEQ ID NO: 30). The V3A hybrid protein comprises, from N-terminus toC-terminus, amino acids 1-226 of a Cry3A055 protein (SEQ ID NO: 70),which comprises variable region 1, conserved block 1, variable region 2,and the N-terminal 34 amino acids of conserved block 2, amino acids237-474 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 33 amino acids of conserved block 2, variable region 3, andthe N-terminal 20 amino acids of conserved block 3, and amino acids467-598 of a Cry3A055 protein (SEQ ID NO: 70), which comprises theC-terminal 28 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, and conserved block 5.

The V3A eHIP comprises two crossover positions. The first crossoverbetween Cry3A055 and Cry1Ab is located in conserved block 2 and thesecond crossover between Cry1Ab and Cry3A055 is located in conservedblock 3. Therefore, V3A is a variant of Cry3A055 in which all ofvariable region 3 has been replaced with variable region 3 of a Cry1Abprotein. The V3A eHIP was not as active against western corn rootworm asFR8a, suggesting that having Cry1Ab sequence in conserved block 3,variable region 4, conserved block 4, variable region 5, conserved block5 and/or variable region 6 is important for full insecticidal activityof FR8a.

The v3A coding sequence was ligated to a pCR2.1-TOPO vector and thensubcloned into pET21a using a BamHI/SacI fragment. The V3A proteinexpressed by the pET21a vector has a T7 tag on the N-terminus. Thisprotein was designated T7-V3A.

Example 18. Construction of V4F

A first nucleic acid fragment encoding variable regions 1-3 of aCry3A055 was PCR amplified from a plasmid comprising cry3A055 (SEQ IDNO: 69) using primers 5′3A-1-bam (SEQ ID NO: 83) and C3-3A-6 (SEQ ID NO:84) and PCR reaction Mix 1 and thermocycling Profile 1.

A second nucleic acid fragment encoding variable region 4 of a Cry1Abwas PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71)using primers C3-1Ab-3 (SEQ ID NO: 85) and C4-3A-10 (SEQ ID NO: 108) andPCR reaction Mix 1 and thermocycling Profile 1.

A third nucleic acid fragment encoding variable regions 5-6 of Cry3A055was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69)using primers C4-3A-9 (SEQ ID NO: 109) and 3A-12-sac (SEQ ID NO: 107)and PCR reaction Mix 1 and thermocycling Profile 1.

All three PCR amplicons were combined and used as the template in anoverlap PCR reaction with primers 5′3A-1-bam (SEQ ID NO: 83) and3A-12-sac (SEQ ID NO: 107) using the following PC conditions: Mix 1 andthermocycling profile: 94° C. —30 seconds, 50° C.-70° C. gradient—30seconds, 72° C.—30 seconds for 20 cycles. The resulting amplicon,designated the v4F coding sequence (SEQ ID NO: 31) which encodes the V4Fhybrid toxin (SEQ ID NO: 32), was cloned into a pCR2.1-TOPO vector anddesignated v4F/pCR2.1. The V4F hybrid protein comprises, from N-terminusto C-terminus, amino acids 1-468 of a Cry3A055 protein (SEQ ID NO: 70),amino acids 477-520, comprising variable region 4, of a Cry1Ab protein(SEQ ID NO: 72), and amino acids 512-598 of a Cry3A055 protein (SEQ IDNO: 70).

The V4F protein has two crossover positions. The first crossover betweenCry3A055 and Cry1Ab is in conserved block 3 and the second crossoverbetween Cry1Ab and Cry3A055 is located in conserved block 4. Therefore,V4F is a variant of Cry3A055 in which all of variable region 4 has beenreplaced with variable region 4 of a Cry1Ab protein. The V4F hybridprotein was not active against western corn rootworm suggesting thatCry1Ab sequence at the C-terminal portion of FR8a contributes to theinsecticidal activity of FR8a.

A BamHI-SacI fragment of v4F/pCR2.1 was subcloned into pET21. Theprotein expressed by the resulting plasmid was designated T7-V4F.

Example 19. Construction of 5*V4F

A BamHI-XbaI fragment from a plasmid comprising FR8a (See Example 4) anda XbaI-SacI fragment from V4F/pCR2.1 (See Example 18) were ligated topET21 cut with BamHI-SacI to form 5*V4F/pET21. The 5*V4F coding sequence(SEQ ID NO: 33) encodes the 5*V4F chimeric protein (SEQ ID NO: 34). The5*V4F chimeric insecticidal protein comprises, from N-terminus toC-terminus, a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-491 of aCry3A055 protein (SEQ ID NO: 70), amino acids 501-520, comprisingvariable region 4, of a Cry1Ab protein (SEQ ID NO: 72), and amino acids512-598 of a Cry3A055 protein (SEQ ID NO: 70).

The 5*V4F eHIP is the V4F hybrid protein with an N-terminal peptidylfragment (SEQ ID NO: 127) added. The 5*V4F eHIP provided insecticidalactivity against western corn rootworm although not at the same level asFR8a. Thus, the N-terminal conferred insecticidal activity to V4Fconfirming that there may be some contributory interaction between theC-terminal portion and the N-terminal peptidyl fragment of FR8a.

The protein expressed by the 5*V4F/pET21 plasmid was designated T7-5*V4Fand has a T7 tag N-terminal to the 5*V4F peptidyl fragment.

Example 20. Construction of 2OL-7

A nucleic acid fragment encoding variable region 1 of Cry3A055 was PCRamplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) usingprimers 5′3A-1-bam (SEQ ID NO: 83) and C1-3A-2 (SEQ ID NO: 92) and PCRreaction Mix 1 and thermocycling Profile 1.

A nucleic acid fragment encoding variable regions 2-6 of Cry1Ab was PCRamplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) usingprimers C1-1Ab-1 (SEQ ID NO: 110) and 1Ab-6-sac (SEQ ID NO: 86) and PCRreaction Mix 1 and thermocycling Profile 1.

The resulting two amplicons were used as templates in an overlap PCRreaction with primers 5′3A-1-bam (SEQ ID NO: 83) and 1Ab-6-sac (SEQ IDNO: 86) using PCR reaction Mix 2 and thermocycling Profile 1, to createthe 2OL-7 coding sequence (SEQ ID NO: 35) which encodes the 2OL-7 hybridprotein (SEQ ID NO: 36). The 2OL-7 hybrid protein comprises, fromN-terminus to C-terminus, amino acids 1-156 of a Cry3A055 protein (SEQID NO: 70), which comprises variable region 1 and the N-terminal 14amino acids of conserved block 1, and amino acids 167-648 of a Cry1Abprotein (SEQ ID NO: 72), which comprises the C-terminal 15 amino acidsof conserved block 1, variable region 2, conserved block 2, variableregion 3, conserved block 3, variable region 4, conserved block 4,variable region 5, conserved block 5 and variable region 6, and 38 aminoacids of the Cry1Ab protoxin tail region Thus, 2OL-7 is a variant of aCry1Ab protein with variable region 1 replaced by variable region 1 froma Cry3A055 protein.

The 2OL-7 coding sequence was cloned into pCR2.1-TOPO (Invitrogen) andthen moved into pET21a using BamHI/SacI which was designated2OL-7/pET21a. The coding sequence in 2OL-7/pET21a was designatedT7-2OL-7 (SEQ ID NO: 37). The protein expressed by the 2OL-7/pET21avector was designated T7-2OL-7 (SEQ ID NO: 38).

Example 21. Construction of 5*2OL-7

A BamHI/XbaI fragment of FR8a (See Example 4), a PpuMI/SacI fragment of2OL-7 (See Example 20) and a BamHI/SacI fragment of pET21a were ligatedto produce 5*2OL-7/pET21a. The 5*2OL-7 coding sequence (SEQ ID NO: 39)encodes the 5*2OL-7 chimeric protein (SEQ ID NO: 40). The 5*2OL-7protein comprises, from N-terminus to C-terminus, a peptidyly fragmentcomprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO:127), amino acids 10-156 of a Cry3A055 protein (SEQ ID NO: 70), andamino acids 167-643 of a Cry1Ab protein (SEQ ID NO: 72). Thus, the5*2OL-7 hybrid protein is the 2OL-7 hybrid protein with a N-terminalpeptidyl fragment added.

Example 22. Construction of 2OL-10

A nucleic acid fragment encoding an N-terminal portion of a Cry3A055protein was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO:69) using primers 5′3A-1-bam (SEQ ID NO: 83) and C2-3A-4 (SEQ ID NO: 88)and PCR reaction Mix 1 and thermocycling Profile 1. A nucleic acidfragment encoding a C-terminal portion of a Cry1Ab protein was PCRamplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) usingprimers C2-3A-3 (SEQ ID NO: 95) and 1Ab-6-sac (SEQ ID NO: 86) and PCRreaction Mix 1 and thermocycling Profile 1. These 2 PCR products werethen used as the templates in an overlap PCR reaction with primers5′3A-1-bam (SEQ ID NO: 83) and 1Ab-6-sac (SEQ ID NO: 86) using thefollowing PCR conditions: Mix 2, thermocycling profile: 94° C.—30seconds, 45° C.-65° C. gradient—30 seconds, 72° C.—30 seconds for 20cycles, resulting in the 2OL-10 coding sequence (SEQ ID NO: 41) whichencodes the 2OL-10 hybrid toxin (SEQ ID NO: 42). The 2OL-10 proteincomprises, from N-terminus to C-terminus, amino acids 1-232 of aCry3A055 protein (SEQ ID NO: 70) and amino acids 243-648 of a Cry1Abprotein (SEQ ID NO: 72). Thus, the 2OL-10 hybrid protein issubstantially Domain I of a Cry3A055 protein and Domains II and III of aCry1Ab protein.

The 2OL-10 coding sequence was cloned into pCR2.1-TOPO (Invitrogen) thenmoved to pET21a using BamHI/SacI. The protein expressed by 2OL-10/pET21awas designated T7-2OL-10.

Example 23. Construction of 5*2OL-10

A BamHI-XbaI fragment from a plasmid comprising FR8a (See Example 4) anda XbaI-SacI fragment from 2OL-10/pCR2.1 (See Example 22) were ligated topET21 cut with BamHI-SacI to form 5*2OL-10/pET21. The 5*2OL-10 codingsequence (SEQ ID NO: 43) encodes the 5*2OL-10 chimeric protein (SEQ IDNO: 44). The 5*2OL-10 protein comprises, from N-terminus to C-terminus,a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-232 of aCry3A055 protein (SEQ ID NO: 70) and amino acids 243-648 of a Cry1Abprotein (SEQ ID NO: 72). Thus, the 5*2OL-10 chimeric protein is the2OL-10 hybrid protein with a N-terminal peptidyl fragment added.

Example 24. Construction of 2OL-12A

A first nucleic acid fragment encoding an N-terminal portion of Cry1Abwas PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71)using primers 5′1Ab-bam (SEQ ID NO: 98) and C3-1Ab-2 (SEQ ID NO: 105)and PCR reaction Mix 1 and thermocycling Profile 1.

A second nucleic acid fragment encoding a C-terminal portion of Cry3A055was PCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69)using primers C3-3A-5 (SEQ ID NO: 106) and 3A-12-sac (SEQ ID NO: 107)and PCR reaction Mix 1 and thermocycling Profile 1.

The first and second nucleic acid fragment described above wereconnected by using them as templates in an overlap PCR reaction withprimers 5′1Ab-bam (SEQ ID NO: 98) and 3A-12-sac (SEQ ID NO: 107) usingMix 1 and thermocycling Profile 1 to create the 2OL-12A coding sequence(SEQ ID NO: 45) which encodes the 2OL-12A eHIP (SEQ ID NO: 46). The2OL-12A protein comprises, from N-terminus to C-terminus, amino acids1-476 of a Cry1Ab protein (SEQ ID NO: 72) and amino acids 469-598 of aCry3A055 protein (SEQ ID NO: 70).

The 2OL-12A eHIP was not active against western corn rootworm but wasactive against European corn borer (See Table 6). This demonstrates thateHIP can be constructed using lepidopteran active and coleopteran activeCry proteins without loss of activity against a lepidopteran insectspecies.

The 2OL-12A coding sequence was cloned into pCR2.1-TOPO (Invitrogen)then moved to pET21a with BamHI/SacI. The protein expressed by the2OL-12A/pET21a vector was designated T7-2OL-12A.

Example 25. Construction of 2OL-13

Four nucleic acid fragments were generated as follows: fragment 1 wasPCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) usingprimers 5′3A-1-bam (SEQ ID NO: 83) and C1-3A-2 (SEQ ID NO: 92) and PCRreaction Mix 1 and thermocycling Profile 1; fragment 2 was PCR amplifiedfrom a plasmid comprising mocry1Ab (SEQ ID NO: 71) using primers C2-3A-3(SEQ ID NO: 95) and C3-1Ab-2 (SEQ ID NO: 105) and PCR reaction Mix 1 andthermocycling Profile 1; fragment 3 was PCR amplified from a plasmidcomprising mocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO:85) and C4-3A-10 (SEQ ID NO: 108) and PCR reaction Mix 1 andthermocycling Profile 1; and fragment 4 was PCR amplified from a plasmidcomprising cry3A055 (SEQ ID NO: 69) using primers C4-3A-9 (SEQ ID NO:109) and 3A-12-sac (SEQ ID NO: 107) and PCR reaction Mix 1 andthermocycling Profile 1.

All four fragments were then used as templates in an overlap PCRreaction using primers 5′3A-bam (SEQ ID NO: 83) and 3A-12-sac (SEQ IDNO: 107) using PCR reaction Mix 1 and thermocycling Profile 1 to createthe 2OL-13 coding sequence (SEQ ID NO: 47) which encodes the 2OL-13hybrid toxin (SEQ ID NO: 48). The 2OL-13 protein comprises, fromN-terminus to C-terminus, amino acids 1-159 of a Cry3A055 protein (SEQID NO: 70), amino acids 170-522 of a Cry1Ab protein (SEQ ID NO: 72), andamino acids 515-598 of a Cry3A055 protein (SEQ ID NO: 70). Thus, the2OL-13 hybrid toxin is comprised of V1 and the N-terminal portion of CB1from a Cry3A055 protein; the C-terminal portion of CB1, V2, CB2, V3,CB3, and V4 from a Cry1Ab protein; and CB4, V5, and CB5 from a Cry3A055protein.

The 2OL-13 coding sequence was cloned into pCR2.1-TOPO (Invitrogen) thenmoved to pET21a using BamHI/SacI. The protein expressed by the2OL-13/pET21a vector was designated T7-2OL-13.

Example 26. Construction of 2OL-20

A BamHI/NspI fragment from a plasmid comprising mocry3A (SEQ ID NO: 67),a NspI/HindIII fragment from a plasmid comprising 2OL-8A (SEQ ID NO: 1),and a HindIII/BamHI fragment from pET21a were ligated to make2OL-20/pET21a.

Example 27. Construction of V5&6

A nucleic acid fragment encoding an N-terminal portion of Cry3A055 wasPCR amplified from a plasmid comprising cry3A055 (SEQ ID NO: 69) usingprimers 5′3A-1-bam (SEQ ID NO: 83) and C4-3A-10 (SEQ ID NO: 108) and PCRreaction Mix 1 and thermocycling Profile 1.

A nucleic acid fragment encoding a C-terminal portion of Cry1Ab was PCRamplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) usingprimers C4-3A-9 (SEQ ID NO: 109) and 1Ab-6-sac (SEQ ID NO: 86) and PCRreaction Mix 1 and thermocycling Profile 1.

These two PCR products were then used as the templates in an overlap PCRreaction with primers 5′3A-1-bam (SEQ ID NO: 83) and 1Ab-6-sac (SEQ IDNO: 86) using PCR reaction Mix 1 and thermocycling Profile 2 to createthe V5&6 coding sequence (SEQ ID NO: 49), which encodes the V5&6 hybridtoxin (SEQ ID NO: 50). The V5&6 protein comprises, from N-terminus toC-terminus, amino acids 1-524 of a Cry3A055 protein (SEQ ID NO: 70),which comprises V1, CB1, V2, CB2, V3, CB3, V4, and CB4, and amino acids533-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises V5, CB5 andV6, and 38 amino acids of a Cry1Ab protoxin tail region.

The V5 &6 coding sequence was cloned into pCR2.1-TOPO then moved topET21 with BamHI/SacI. The protein expressed by V5&6/pET21a wasdesignated T7-V5&6.

Example 28. Construction of 5*V5&6

A BamHI/XbaI fragment of FR8a (See Example 4), a XbaI/SacI fragment ofV5 &6 (See Example 27) and a BamHI/SacI fragment of pET21a were ligatedto form 5*V5&6/pET21. The 5*V5&6 coding sequence (SEQ ID NO: 51) encodesthe 5*V5&6 chimeric protein (SEQ ID NO: 52). The 5*V5&6 chimericinsecticidal protein comprises, from N-terminus to C-terminus, apeptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-524 of aCry3A055 protein (SEQ ID NO: 70), which comprises V1, CB1, V2, CB2, V3,CB3, V4, and CB4, and amino acids 533-648 of a Cry1Ab protein (SEQ IDNO: 72), which comprises V5, CB5 and V6, and 38 amino acids of a Cry1Abprotoxin tail region. Thus, the 5*V5&6 chimeric insecticidal protein isthe V5&6 hybrid protein with an N-terminal peptidyl fragment added.

Example 29. Construction of 88A-dm3

A nucleic acid fragment encoding a C-terminal portion of a Cry8Aaprotein (SEQ ID NO: 78) was PCR amplified from a plasmid comprisingcry8Aa (SEQ ID NO: 77) using primers 5′8Aa-dm3 (SEQ ID NO: 111) and3′8Aa-dm3 (SEQ ID NO: 112) and PCR reaction Mix 2 and thermocyclingProfile 2. The resulting amplicon was cloned into pCR2.1-TOPO(Invitrogen) and designated 88A-dm3/pCR2.1.

A MluI/SacI fragment from 88A-dm3/pCR2.1 and a SacI/MluI fragment from aplasmid comprising FR8a (See Example 4) were ligated to create the88A-dm3 coding sequence (SEQ ID NO: 53) which encodes the 88A-dm3 hybridprotein (SEQ ID NO: 54). The 88A-dm3 protein comprises, from N-terminusto C-terminus, a peptidyl fragment comprising the amino acid sequenceMTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 of aCry3A055 protein (SEQ ID NO: 70) and amino acids 532-664 of a Cry8Aaprotein (SEQ ID NO: 78).

The 88A-dm3 coding sequence was also transformed into pET21a using aBamHI/SacI restriction digest and ligation. The protein expressed by88A-dm3/pET21a was designated T7-88A-dm3.

Example 30. Construction of FR(1Fa)

A nucleic acid fragment encoding an N-terminal portion of FR8a (SeeExample 3) was PCR amplified from a plasmid comprising FR8a (SEQ IDNO: 1) using primers C2-3A-3 (SEQ ID NO: 95) and tant-OL-2 (SEQ ID NO:113) and PCR reaction Mix 3 and thermocycling Profile 3.

A nucleic acid fragment encoding a C-terminal portion of a Cry1Faprotein (SEQ ID NO: 76) was PCR amplified from a plasmid comprisingmocry1Fa (SEQ ID NO: 75) using primers tant-OL-1 (SEQ ID NO: 114) andtant-3′sac (SEQ ID NO: 115) and PCR reaction Mix 3 and thermocyclingProfile 3.

These two PCR products were then used as templates in an overlap PCRreaction with primers C2-3A-3 (SEQ ID NO: 95) and tant-3′sac (SEQ ID NO:115) using PCR reaction Mix 3 and thermocycling Profile 3. The resultingPCR product was cloned into pCR2.1-TOPO (Invitrogen). A BamHI/MluIfragment from a plasmid comprising FR8a, a MluI/SacI fragment from theoverlap PCR product in pCR2.1 and a BamHI/SacI fragment of pET21a werethen ligated to create FR(1Fa)/pET21a. The FR(1Fa) coding sequence (SEQID NO: 55) encodes the FR(1Fa) chimeric protein (SEQ ID NO: 56). TheFR(1Fa) protein comprises, from N-terminus to C-terminus, a peptidylfragment comprising the amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQID NO: 127), amino acids 10-468 of a Cry3A055 protein (SEQ ID NO: 70)and amino acids 470-649 of a Cry1Fa protein (SEQ ID NO: 76).

Example 31. Construction of FR(1Ac)

Domains I & II of FR8a were PCR amplified from a plasmid comprising FR8a(SEQ ID NO: 1) using primers C2-3A-3 (SEQ ID NO: 95) and 1Ac-OL-2 (SEQID NO: 116) and PCR reaction Mix 3 and thermocycling Profile 3. DomainIII of Cry1Ac (SEQ ID NO: 80) was PCR amplified from a plasmidcomprising cry1Ac (SEQ ID NO: 79) using primers 1Ac-OL-1 (SEQ ID NO:117) and 1Ac-3′sac (SEQ ID NO: 118) and PCR reaction Mix 3 andthermocycling Profile 3.

These 2 PCR products were used as templates in an overlap PCR reactionwith primers C2-3A-3 (SEQ ID NO: 95) and 1Ac-3′sac (SEQ ID NO: 118) andthe following conditions: Mix 3 and thermocycling profile: 94° C.—30seconds, 68° C.—30 seconds, 68° C.—30 seconds for 20 cycles. The overlapPCR product was cloned into pCR2.1-TOPO (Invitrogen). A BamHI/MluIfragment from a plasmid comprising FR8a, the MluI/SacI fragment from theoverlap PCR product in pCR2.1 and BamHI/SacI fragment of pET21a wereligated to create FR(1Ac)/pET21a. The FR(1Ac) protein comprises, fromN-terminus to C-terminus, a peptidyl fragment comprising the amino acidsequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-468 ofa Cry3A055 protein (SEQ ID NO: 70) and amino acids 477-608 of a Cry1Acprotein (SEQ ID NO: 80).

Example 32. Construction of FR(1Ia)

A nucleotide fragment encoding Domains I and II of FR8a was PCRamplified from a plasmid comprising FR8a (SEQ ID NO: 3) using primersC2-3A-3 (SEQ ID NO: 95) and 1Ia-OL-2 (SEQ ID NO: 119) and PCR reactionMix 3 and thermocycling Profile 3. A second nucleotide fragment encodingDomain III of a Cry1Ia protein (SEQ ID NO: 82) was PCR amplified from aplasmid comprising cry1Ia (SEQ ID NO: 81) using primers 1Ia-OL-1 (SEQ IDNO: 120) and 1Ia-3′sac (SEQ ID NO: 121) and PCR reaction Mix 3 andthermocycling Profile 3. These two PCR products were used as templatesin an overlap PCR reaction with primers C2-3A-3 (SEQ ID NO: 95) and1Ia-3′sac (SEQ ID NO: 121) and PCR reaction Mix 3 and thermocyclingprofile: 94° C.—30 seconds, 68° C. —45 seconds for 20 cycles. Theoverlap PCR product was cloned into pCR2.1-TOPO (Invitrogen). TheBamHI/MluI fragment from a plasmid comprising FR8a, the MluI/SacIfragment from the overlap PCR product in pCR2.1 and BamHI/SacI fragmentof pET21a were ligated to create FR(1Ia)/pET21a. The FR(1Ia) proteincomprises, from N-terminus to C-terminus, a peptidyl fragment comprisingthe amino acid sequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), aminoacids 10-468 of a Cry3A055 protein (SEQ ID NO: 70) and amino acids513-719 of a Cry1Ia protein (SEQ ID NO: 82).

Example 33. Construction of Dm2-3A

Part of the 5′ end of this coding sequence was PCR amplified from aplasmid comprising cry3A055 (SEQ ID NO: 69) using primers C2-3A-3 (SEQID NO: 95) and FR-1Ab-2 (SEQ ID NO: 122) and PCR reaction Mix 3 andthermocycling Profile 2. A nucleotide fragment encoding Domain III ofCry1Ab was PCR amplified from a plasmid comprising mocry1Ab (SEQ ID NO:71) using primers FR1Ab-1 (SEQ ID NO: 123) and 1Ab-6-sac (SEQ ID NO: 86)and PCR reaction Mix 3 and thermocycling Profile 2. These two PCRproducts were used as the templates in an overlap PCR reaction withprimers C2-3A-3 (SEQ ID NO: 95) and 1Ab-6-sac (SEQ ID NO: 86) and PCRreaction Mix 3 and thermocycling Profile 2. The resulting amplicon wascloned into pCR2.1-TOPO (Invitrogen). FR8a BamHI/MluI, and the above PCRproduct in pCR2.1-TOPO AflIII, FR8a AflIII/SacI were ligated into pET21aBamHI/SacI. The entire coding sequence (BamHI/SacI) was then moved to1454. The DM2-3A chimeric insecticidal protein comprises, fromN-terminus to C-terminus, a peptidyl fragment comprising the amino acidsequence MTSNGRQCAGIRPYDGRQQHRG (SEQ ID NO: 127), amino acids 10-451 ofa Cry3A055 protein (SEQ ID NO: 70), which comprises variable region 1,conserved block 1, variable region 2, conserved block 2, variable region3, and the N-terminal 7 amino acids of conserved block 3, and aminoacids 460-648 of a Cry1Ab protein (SEQ ID NO: 72), which comprises theC-terminal 41 amino acids of conserved block 3, variable region 4,conserved block 4, variable region 5, conserved block 5, and variableregion 6. Thus, the DM2-3A eHIP has a cross-over junction betweenCry3A055 and Cry1Ab located in conserved block 3 immediately followingSer451 which is upstream of the domain II domain III junction. DM2-3Ahas insecticidal activity against western corn rootworm but the activitywas less than that of the 8AF and FR8a eHIPs as shown in Table 5.

Example 34. Construction of T7-8AF

A nucleic acid fragment encoding an N-terminal portion of a Cry3A055protein (SEQ ID NO: 70) was PCR amplified from a plasmid comprisingcry3A055 (SEQ ID NO: 69) using primers 5′3A-1-bam (SEQ ID NO: 83) andC3-3A-6 (SEQ ID NO: 84) and PCR reaction Mix 1 and thermocycling Profile1.

A nucleic acid fragment encoding a C-terminal portion of a Cry1Abprotein (SEQ ID NO: 72) was PCR amplified from a plasmid comprisingmocry1Ab (SEQ ID NO: 71) using primers C3-1Ab-3 (SEQ ID NO: 85) and1Ab-6-Sac (SEQ ID NO: 86) and PCR reaction Mix 1 and thermocycle Profile1.

The two above-described PCR products were next used as templates in anoverlap PCR reaction with the primers 5′3A-1-bam (SEQ ID NO: 83) and1Ab-6-Sac (SEQ ID NO: 86) using PCR reaction Mix 2 and thermocyclingProfile 1.

The resulting amplicon was ligated as a blunt ended fragment to apCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.) cut with Smal to formplasmid p8AF/CR2.1. A BamHI-SacI fragment from p8AF/CR2.1 was thenligated to pET21a (EMD Biosciences, Inc., San Diego, Calif.), which wascut with BamHI-SacI, and transformed into E. coli. The open readingframe was designated T7-8AF (SEQ ID NO: 144) and encodes the T7-8AFhybrid protein (SEQ ID NO: 145). The T7-8AF hybrid protein comprises,from N-terminus to C-terminus, a peptidyl fragment comprising the aminoacid sequence MASMTGGQQMGRGS (amino acids 1-14 of SEQ ID NO: 126), aminoacids 1-468 of a Cry3A055 protein (SEQ ID NO: 70), which comprisesvariable region 1, conserved block 1, variable region 2, conserved block2, variable region 3, and the N-terminal 24 amino acids of conservedblock 3, and amino acids 477-648 of a Cry1Ab protein (SEQ ID NO: 72),which comprises the C-terminal 24 amino acids of conserved block 3,variable region 4, conserved block 4, variable region 5, conserved block5 and variable region 6, and a 38 amino acid region of the Cry1Abprotoxin tail. The T7-8AF hybrid protein had little or no insecticidalactivity against western corn rootworm.

Example 35. Construction of 8AF

A BamHI-SacI fragment from plasmid p8AF/CR2.1 (See Example 34) wasligated to a plasmid containing a constitutive Cry1Ac promoter that hasbeen modified from that described by Schnepf et al. (1985. J. Biol.Chem. 260:6264-6272) to correct an internal ATG start codon which existsin the promoter of Schnepf et al. to an ATC codon, which was cut withBamHI-SacI, and transformed into E. coli. The open reading frame wasdesignated 8AF (SEQ ID NO: 63) and encodes the 8AF eHIP (SEQ ID NO: 64).The 8AF eHIP is similar to the FR8a eHIP but does not contain theoptional N-terminal peptidyl fragment. The 8AF eHIP comprises, fromN-terminus to C-terminus, amino acids 1-468 of a Cry3A055 protein (SEQID NO: 70), which comprises variable region 1, conserved block 1,variable region 2, conserved block 2, variable region 3, and theN-terminal 24 amino acids of conserved block 3, and amino acids 477-648of a Cry1Ab protein (SEQ ID NO: 72), which comprises the C-terminal 24amino acids of conserved block 3, variable region 4, conserved block 4,variable region 5, conserved block 5 and variable region 6, and a 38amino acid region of a Cry1Ab protoxin tail. Thus, the 8AF eHIP has across-over junction between Cry3A055 and Cry1Ab located in conservedblock 3 immediately following Leu468 of SEQ ID NO: 70 which isdownstream of the domain II domain III junction. The 8AF eHIP had highactivity against western corn rootworm.

Example 36. Construction of −CatG8AF

A construct was made without the Cathepsin G (Cat G) site to determinewhether the Cat G site in domain I of the 8AF eHIP was necessary forrootworm activity. A 1359 bp BamHI/SalI fragment from a plasmidcomprising moCry3A (SEQ ID NO: 67) and a 3483 bp BamHI/SalI fragmentfrom a plasmid comprising 2OL-8a (SEQ ID NO: 1) were ligated to create−catG8AF (SEQ ID NO: 146) which encodes the −catG8AF eHIP (SEQ ID NO:147).

The −catG8AF eHIP was very active against western corn rootwormdemonstrating that the Cathepsin G protease recognition site in the 8AFeHIP is not required for insecticidal activity.

Example 37. Construction of 8AFdm3

The 8AF eHIP described in Example 35 has a cross-over point betweenCry3A055 and Cry1Ab located in CB3 downstream of the domain II/IIIjunction, resulting in domain III of the 8AF eHIP having a smallN-terminal region of domain III of Cry3A055 and the remainder of domainIII being Cry1Ab domain III sequence. To determine whether the smallN-terminal region of domain III of Cry3A055 was required forinsecticidal activity in 8AF, another construct was made having thecross-over between Cry3A055 and Cry1Ab located in CB3 exactly at thedomain II-domain III junction.

A nucleic acid fragment encoding part of domain I and domain II ofCry3A055 was PCR amplified from a plasmid comprising FR8a (SEQ ID NO: 3)using primers CMS96 (SEQ ID NO: 138) and CMS97 (SEQ ID NO: 139) and PCRreaction Mix 5 and thermocycle Profile 5.

A nucleic acid fragment encoding domain III of moCry1Ab was PCRamplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) usingprimers CMS98 (SEQ ID NO: 140) and CMS99 (SEQ ID NO: 141) and PCRreaction Mix 5 and thermocycle Profile 5.

The resulting two amplicons were used as templates in an overlap PCRreaction with primers CMS96 (SEQ ID NO: 138) and CMS98 (SEQ ID NO: 140)using PCR reaction Mix 5 and thermocycle Profile 6. The resultingamplicon was cloned into pCR4 Blunt (Invitrogen, Carlsbad, Calif.). A1633 bp StuI/SacI fragment of the cloned amplicon, designatedpCR4Blunt-OLWrdm3, and a approximately 3089 bp StuI/SacI fragment of aplasmid comprising cry3A055 (SEQ ID NO: 69) were combined to create8AFdm3 (SEQ ID NO: 148) which encodes the 8AFdm3 hybrid protein (SEQ IDNO: 149).

The 8AFdm3 hybrid protein comprises, from N-terminus to C-terminus,amino acids 1-454 of a Cry3A055 protein (SEQ ID NO: 70), which comprisesdomains I and II, which comprise variable region 1, conserved block 1,variable region 2, conserved block 2, variable region 3, and theN-terminal 10 amino acids of conserved block 3, and amino acids 463-610of a Cry1Ab protein (SEQ ID NO: 72), which comprises all of domain III,comprising the C-terminal 38 amino acids of conserved block 3, variableregion 4, conserved block 4, variable region 5, conserved block 5 andvariable region 6.

Thus, the 8AFdm3 protein has a cross-over junction between Cry3A055 andCry1Ab immediately after Phe454 of SEQ ID NO: 70, which is at the domainII-domain III junction. The 8AFdm3 protein had no activity againstwestern corn rootworm. This suggests that the 24 amino acid N-terminalregion of CB3 of Cry3A055 or Cry3A, since they have the same sequence inthis region, are necessary for activity of an 8AF eHIP.

Example 38. Construction of 8AFlongdm3

To determine if the location of the cross-over junction in CB3 betweenCry3A or Cry3A005 and Cry1Ab was critical for rootworm activity aconstruct was made wherein the cross-over junction was placed in CB4immediately after amino acid 519 of a Cry3A055 protein.

A nucleic acid fragment encoding part of domain I and all of domain IIand part of domain III of Cry3A055 was PCR amplified from a plasmidcomprising cry3A055 (SEQ ID NO: 69) using primers CMS96 (SEQ ID NO: 138)and CMS101 (SEQ ID NO: 143) and PCR reaction Mix 5 and thermocycleProfile 5.

A nucleic acid fragment encoding part of domain III of Cry1Ab was PCRamplified from a plasmid comprising mocry1Ab (SEQ ID NO: 71) usingprimers CMS98 (SEQ ID NO: 140) and CMS100 (SEQ ID NO: 142) and PCRreaction Mix 5 and thermocycle Profile 5.

The resulting two amplicons were used as templates in an overlap PCRreaction with primers CMS96 (SEQ ID NO: 138) and CMS98 (SEQ ID NO: 140)using PCR reaction Mix 5 and thermocycle Profile 6. The resultingamplicon was cloned into pCR4 Blunt (Invitrogen, Carlsbad, Calif.). Aapproximately 460 bp SalI/SacI fragment of the cloned amplicon,designated pCR4Blunt-OL8AFlongdm3, and a approximately 4265 bp SalI/SacIfragment of a plasmid comprising 8AFdm3 (SEQ ID NO: 147) were combinedto create 8AFlongdm3 (SEQ ID NO: 150) which encodes the 8AFlongdm3hybrid protein (SEQ ID NO: 151).

The 8AFlongdm3 hybrid protein comprises, from N-terminus to C-terminus,amino acids 1-519 of a Cry3A055 protein (SEQ ID NO: 70), which comprisesdomains I and II, which comprise variable region 1, conserved block 1,variable region 2, conserved block 2, variable region 3, conserved block3, variable region 4, and the N-terminal 6 amino acids of conservedblock 4, and amino acids 528-610 of a Cry1Ab protein (SEQ ID NO: 72),which comprises a C-terminal region of domain III, comprising theC-terminal 4 amino acids of conserved block 4, variable region 5,conserved block 5, and variable region 6.

Thus, the 8AFlongdm3 protein has a cross-over junction between Cry3A055and Cry1Ab in conserved block 4 immediately after Ile519 of SEQ ID NO:70. The 8AFlongdm3 hybrid Cry protein had no activity against westerncorn rootworm. This suggests that a critical region for corn rootwormactivity of a Cry3A-Cry1A eHIP lies in a region between amino acidscorresponding to amino acid 6 of CB3 to amino acid 7 of CB4.

Example 39. Construction of Cap8AFdm3

A approximately 1363 bp BamHI/SalI fragment from a plasmid comprising8AFdm3 (SEQ ID NO: 148) and a approximately 3362 bp BamHI/SalI fragmentfrom a plasmid comprising FR8a (SEQ ID NO: 3) were ligated to createcap8AFdm3 (SEQ ID NO: 152) which encodes the cap8AFdm3 eHIP (SEQ ID NO:153).

The cap8AFdm3 protein had some activity against western corn rootworm asindicated in Table 5. The only difference between the 8AFdm3 hybridprotein, which was not insecticidal, and the cap8AFdm3 eHIP is thepresence of an N-terminal peptidyl fragment (SEQ ID NO: 127). Thus,adding a peptidyl fragment to a non-active hybrid Cry protein created arootworm active engineered hybrid insecticidal protein.

Example 40. Construction of 8AFdm3T

A approximately 4654 bp PmlI/SacI fragment from a plasmid comprising8AFdm3 (SEQ ID NO: 148) and a approximately 190 bp PmlI/SacI fragmentfrom a plasmid comprising FR8a (SEQ ID NO: 3) were ligated to create8AFdm3T (SEQ ID NO: 154) which encodes the 8AFdm3T eHIP (SEQ ID NO:155). The 8AFdm3T eHIP comprises from N-terminus to C-terminus, aminoacids 1-454 of a Cry3A055 protein (SEQ ID NO: 70), which comprisesdomains I and II, which comprise variable region 1, conserved block 1,variable region 2, conserved block 2, variable region 3, and theN-terminal 10 amino acids of conserved block 3, and amino acids 463-610of a Cry1Ab protein (SEQ ID NO: 72), which comprises all of domain III,comprising the C-terminal 38 amino acids of conserved block 3, variableregion 4, conserved block 4, variable region 5, conserved block 5,variable region 6, and a 38 amino acid region of a Cry1Ab protoxin tail.

The only difference between the 8AFdm3 hybrid protein and the 8AFdm3TeHIP is the addition of the 38 amino acid Cry1Ab protoxin tail regionindicating that addition of a protoxin tail region can change anon-active hybrid Cry protein into an active eHIP.

Example 41. Construction of 8AFlongdm3T

A approximately 4693 bp PmlI/SacI fragment from a plasmid comprising8AFlongdm3 (SEQ ID NO: 150) and a approximately 190 bp PmlI/SacIfragment from a plasmid comprising FR8a (SEQ ID NO: 3) were ligated tocreate 8AFlongdm3T (SEQ ID NO: 156) which encodes the 8AFlongdmT hybridCry protein (SEQ ID NO: 157).

The only difference between the 8AFlongdm3 hybrid Cry protein and the8AFlongdm3T hybrid Cry protein, which was not active against westerncorn rootworm, is the addition of a 38 amino acid Cry1Ab protoxin tailregion indicating that the protoxin region was not itself sufficient toconfer insecticidal activity to the 8AFlongdm3 hybrid Cry protein. Thisindicates that a combination of variable regions and conserved blocks inaddition to a protoxin tail region and/or an N-terminal peptidylfragment may be necessary to create some eHIPs.

Example 42. Construction of Cap8AFdm3 T

A approximately 4693 bp PmlI/SacI fragment from a plasmid comprisingcap8AFdm3 (SEQ ID NO: 152) and a approximately 190 bp PmlI/SacI fragmentfrom a plasmid comprising FR8A (SEQ ID NO: 3) were ligated to createcap8AFdm3T (SEQ ID NO: 158) which encodes the cap8AFdm3T eHIP (SEQ IDNO: 159).

The cap8AFdm3T protein had increased activity against western cornrootworm over the cap8AFdm3 eHIP as indicated in Table 5. The onlydifference between the cap8AFdm3 eHIP, which had some insecticidalactivity against corn rootworm, and the cap8AFdm3T eHIP is the presenceof a 38 amino acid protoxin tail region from Cry1Ab. Thus, some hybridCry proteins can be made active by adding an N-terminal peptidylfragment and a protoxin tail region.

Example 43. Testing Hybrid Proteins for Insecticidal Activity

Western Corn Rootworm

Hybrid proteins generated in the above described Examples were testedfor insecticidal activity against western corn rootworm in laboratorybioassays. Bioassays were performed using a diet incorporation method.E. coli clones that express one of the proteins were grown overnight.500 μl of an overnight culture was sonicated and the amount of proteinto be tested was determined. The protein solution was then mixed with500 μl of molten artificial diet similar to that described in Marrone etal. (1985, J. of Economic Entomology 78:290-293). After the dietsolidified, it was dispensed in a petri-dish and 20 neonate cornrootworm were placed on the diet. The petri-dishes were held atapproximately 30° C. Mortality was recorded after 6 days.

Results of the bioassays are shown in Table 5. Column 1 indicates thenames of the hybrid Cry proteins, engineered hybrid insecticidalproteins and chimeric insecticidal proteins. Column 2 indicates relativelevels of western corn rootworm activity (“−”=<40% mortality; “+”=40-49%mortality; “++”=50-59% mortality; “+++”=60-80% mortality; and“++++”=>80% mortality). Column 3 indicates relative levels of theappropriate protein detected by Western blot. Column 4 indicatespresence of a peptidyl fragment (“−”=No peptidyl fragment; #1=SEQ ID NO:126; #2=SEQ ID NO: 127; #3=SEQ ID NO: 128; #4=SEQ ID NO: 129; #5=SEQ IDNO: 130; #6=SEQ ID NO: 131; #7=SEQ ID NO: 132). Columns 5-7 show thecombinations and arrangement of the variable regions (V1-V6), conservedblocks (C1-C5) and associated domains (Domain I-III) from a first Bt Cryprotein or modified Cry protein and a second Bt Cry protein differentfrom the first Cry protein or modified Cry protein that make up a corehybrid protein, which are not active against western corn rootworm andeHIPs, which have activity against western corn rootworm. Column 8indicates the number of amino acids in a protoxin tail region if presentand the Cry protein from which the tail region is derived (“1Ab-38”=38amino acids from a Cry1Ab protoxin tail; “1Ba-18”=18 amino acids from aCry1Ba protoxin tail).

TABLE 5 Results of western corn rootworm bioassays. Domain Domain DomainProteins CRW Protein Peptidyl I II III Protoxin Tested ActivityExpressed Fragment V1 C1 V2 C2 V3 C3 V4 C4 V5 C5 V6 Region 8AF ++++ ++ —3A055 3A055 3A055 1Ab 1Ab-38 T7-8AF − + #7 3A055 3A055 3A055 1Ab 1Ab-38-CatG8AF ++++ ++ — 3A 3A 3A 1Ab 1Ab-38 8AFdm3 − + — 3A055 3A055 1Ab —8AFdm3T +++ ++ — 3A055 3A055 1Ab 1Ab-38 8AFlongdm3 − + — 3A055 3A0553A055 1Ab — 8AFlongdm3T − + — 3A055 3A055 3A055 1Ab 1Ab-38 Cap8AFdm3 + +#2 3A055 3A055 1Ab — Cap8AFdm3T ++ ++ #2 3A055 3A055 1Ab 1Ab-38 2OL-8a++++ ++ #1 3A055 3A055 3A055 1Ab 1Ab-38 FR8a +34 ++++ ++ #6 3A055 3A0553A055 1Ab 1Ab-38 FR8a ++++ ++ #2 3A055 3A055 3A055 1Ab 1Ab-38 FRCG ++++++ #2 3A 3A 3A 1Ab 1Ab-38 FR8a-9F +++ ++ #5 3A055 3A055 3A055 1Ab 1Ab-38FR8a-9F-catg ++++ ++ #5 3A 3A 3A 1Ab 1Ab-38 FR8a-12aa ++++ ++ #3 3A0553A055 3A055 1Ab 1Ab-38 Cry3A055 ++++ ++ — 3A055 3A055 3A055 — 5*Cry3A055− ++ #2 3A055 3A055 3A055 — Wr-9mut − ++ #3 3A055 3A055 3A055 — FRD3++++ ++ #2 3A055 3A055 3A055 1Ab — FR-12-cg-dm3 ++ ++ #3 3A055 3A0553A055 1Ab — 9F-cg-del6 − ++ #5 3A 3A 3A 1Ab 1Ab-38 FR-cg-dm3 ++++ ++ #23A 3A 3A 1Ab — 9F-cg-dm3 ++++ ++ #5 3A 3A 3A 1Ab — B8a − + — 3A055 3A0553A055 1Ba 1Ba-18 5*B8a − + #2 3A055 3A055 3A055 1Ba 1Ba-18 V3A ++ + —3A055 1Ab 3A055 — V4F − ++ — 3A055 3A055 1Ab 3A055 — 5*V4F ++ + #2 3A0553A055 1Ab 3A055 — 2OL-7 − ++ — 3A055 1Ab 1Ab 1Ab 1Ab-38 5*2OL-7 − + #23A055 1Ab 1Ab 1Ab 1Ab-38 2OL-10 − + — 3A055 1Ab 1Ab 1Ab-38 5*2OL-10 +/−+/− #2 3A055 1Ab 1Ab 1Ab-38 2OL-12A − ++ — 1Ab 1Ab 3A — 2OL-13 − − —3A055 1Ab 1Ab 3A055 — 2OL-20 − + — 3A 3A 3A 1Ab 1Ab-38 V5&6 − ++ — 3A0553A055 3A055 1Ab 1Ab-38 5*V5&6 − ++ #2 3A055 3A055 3A055 1Ab 1Ab-3888A-dm3 − ++ #2 3A055 3A055 3A055 8Aa — FR(1Fa) − ++ #2 3A055 3A0553A055 1Fa — FR(1Ac) − + #2 3A055 3A055 3A055 1Ac — FR(1Ia) − − #2 3A0553A055 3A055 1Ia — DM23A + + #2 3A055 3A055 3A055 1Ab 1Ab-38

The chimeric insecticidal proteins, 2OL-8a and FR8a, and the 2OL-12AeHIP, were tested against several insect species to determine spectrumof activity. The insects tested included western corn rootworm (WCR),northern corn rootworm (NCR), southern corn rootworm (SCR), Coloradopotato beetle (CPB), and European corn borer (ECB). Results of theassays are shown in Table 6. A “+” indicates insecticidal activity. A“−” indicates no activity. The 2OL-8a and FR8a CIPs were active againstWCR, NCR and CPB. The 2OL-12A eHIP was surprisingly active against ECB.

TABLE 6 Activity spectrum of CIPs. Activity Spectrum Protein WCR NCR SCRCPB ECB 2OL-8a + + − + − FR8a + + − + − 2OL-12A − nt nt nt +Cry3A055 + + − + − Cry3A − − − + − Cry1Ab − − − − +

Example 44. Insertion of Genes Encoding eHIPs into Plants

Three genes encoding the chimeric insecticidal proteins FR8a, FRCG andFRD3 were chosen for transformation into maize plants. An expressioncassette comprising the FR8a or FRCG or FRD3 coding sequence wastransferred to a suitable vector for Agrobacterium-mediated maizetransformation. For this example, the following vectors were used in thetransformation experiments: 12207 (FIG. 3), 12161 (FIG. 4), 12208 (FIG.5), 12274 (FIG. 6), 12473 (FIG. 7) and 12474 (FIG. 8).

Transformation of immature maize embryos was performed essentially asdescribed in Negrotto et al., 2000, Plant Cell Reports 19: 798-803. Forthis example, all media constituents were essentially as described inNegrotto et al., supra. However, various media constituents known in theart may be substituted.

The genes used for transformation were cloned into a vector suitable formaize transformation. Vectors used in this example contain thephosphomannose isomerase (PMI) gene for selection of transgenic lines(Negrotto et al., supra).

Briefly, Agrobacterium strain LBA4404 (pSB 1) containing a planttransformation plasmid was grown on YEP (yeast extract (5 g/L), peptone(10 g/L), NaCl (5 g/L), 15 g/l agar, pH 6.8) solid medium for 2-4 daysat 28° C. Approximately 0.8×10⁹ Agrobacterium were suspended in LS-infmedia supplemented with 100 μM As (Negrotto et al., supra). Bacteriawere pre-induced in this medium for 30-60 minutes.

Immature embryos from A188 or other suitable genotype are excised from8-12 day old ears into liquid LS-inf+100 μM As. Embryos are rinsed oncewith fresh infection medium. Agrobacterium solution is then added andembryos are vortexed for 30 seconds and allowed to settle with thebacteria for 5 minutes. The embryos are then transferred scutellum sideup to LSAs medium and cultured in the dark for two to three days.Subsequently, between 20 and 25 embryos per petri plate are transferredto LSDc medium supplemented with cefotaxime (250 mg/1) and silvernitrate (1.6 mg/1) and cultured in the dark for 28° C. for 10 days.

Immature embryos, producing embryogenic callus were transferred toLSD1M0.5S medium. The cultures were selected on this medium for about 6weeks with a subculture step at about 3 weeks. Surviving calli weretransferred to Reg1 medium supplemented with mannose. Followingculturing in the light (16 hour light/8 hour dark regiment), greentissues were then transferred to Reg2 medium without growth regulatorsand incubated for about 1-2 weeks. Plantlets were transferred to MagentaGA-7 boxes (Magenta Corp, Chicago Ill.) containing Reg3 medium and grownin the light. After about 2-3 weeks, plants were tested for the presenceof the pmi gene and the FR8a or FRCG genes by PCR. Positive plants fromthe PCR assay were transferred to the greenhouse and tested forresistance to corn rootworm.

Example 45. Analysis of Transgenic Maize Plants for Corn RootwormEfficacy: Root Excision Bioassay

Typically, corn plants are sampled as they are being transplanted fromMagenta GA-7 boxes into soil. This allows the roots to be sampled from areasonably sterile environment relative to soil conditions. Samplingconsists of cutting a small piece of root (ca. 2-4 cm long) and placingit onto enriched phytagar (phytagar, 12 g., sucrose, 9 g., MS salts, 3ml., MS vitamins, 3 ml., Nystatin (25 mg/ml), 3 ml., Cefotaxime (50mg/ml), 7 ml., Aureomycin (50 mg/ml), 7 ml., Streptomycin (50 mg/ml), 7ml., dH₂O, 600 ml) in a small petri-dish. Negative controls are eithertransgenic plants that are PCR negative for the FR8a or FRCG gene fromthe same transformation experiment, or from non-transgenic plants (of asimilar size to test plants) that were being grown in the phytotron.

Roots are also sampled after plants have been growing in soil. Ifsampling roots from soil, the root pieces are washed with water toremove soil residue, dipped in Nystatin solution (5 mg/ml), removed fromthe dip, blotted dry with paper toweling, and placed into a phytagardish as above.

Root samples are inoculated with western corn rootworms by placing about10 first instar larvae onto the inside surface of the lid of eachphytagar dish and the lids then tightly resealed over the exposed rootpiece. Larvae are handled using a fine tip paintbrush. After all disheswere inoculated, the tray of dishes was placed in the dark at roomtemperature until data collection.

At about 2-4 days after root inoculation, data were collected. Thepercent mortality of the larvae was calculated along with a visualdamage rating of the root. Feeding damage was scored by observing thenumber of feeding holes (FH) in the root piece caused by the rootwormlarvae and was rated as high, moderate, low, or absent and given anumerical value of category 3, 2, or 1, respectively (with Category 1including damage ratings of absent and/or low). Category 1 plantstypically have 0-FH to 2-FH; Category 2 plants have 3 to 4-FH; andCategory 3 plants have >5-FH. Root samples having a damage rating inCategory 1 were considered excellent performers, category 2: averageperformers and category 3: poor performers. Category 1 plants wereselected for further testing in the greenhouse and field.

Results in Table 7 show that plants expressing a the FR8a and FRCG eHIPsprotected roots from feeding damage caused by western corn rootworm. Amajority of events expressing the chimeric insecticidal protein wereconsidered category 1 plants, whereas control plants not expressing achimeric insecticidal protein were in category 3. Plants expressing theFRD3 eHIP provided comparable levels of control of western cornrootworm.

TABLE 7 Efficacy of transgenic plants expressing FR8a and FRCG againstWCR. Damage Rating Vector Event (No. FH) Category 12161 1 1 1 2 1 1 3 01 4 3 2 5 2 1 6 0 1 7 0 1 8 0 1 9 1 1 10 1 1 11 4 2 12 0 1 Control 1 6 32 6 3 3 6 3 4 18 3 12208 1 0 1 2 0 1 3 3 2 4 4 2 5 1 1 6 4 2 7 0 1 8 4 29 4 2 10 1 1 11 1 1 12 1 1 13 0 1 Control 1 10 3 2 5 3 3 7 3 4 7 3 5 8 36 8 3 12207 1 0 1 2 2 1 3 1 1 4 2 1 5 1 1 6 2 1 7 4 2 8 3 2 9 4 2Control 1 7 3 2 9 3 3 8 3 4 11 3 5 7 3 6 12 3 12274 1 3 2 2 0 1 3 3 2 43 2 5 0 1 6 3 2 7 3 2 8 0 1 9 3 2 10 3 2 11 0 1 Control 1 10 3 2 10 3 310 3 4 7 3 5 8 3 6 6 3

Example 46. Analysis of Transgenic Maize Plants for Corn RootwormEfficacy in the Field

Some positive plants identified using the root excision bioassaydescribed above were evaluated in the field. Eighteen plants from eachevent were removed from field plots and evaluated for damage to theroots. Root damage was rated using the Iowa State 0 to 3 linear rootdamage scale (Oleson, J. D. et al., 2005. J. Econ Entomol. 98(1): 1-8),where 0.00=no feeding damage (lowest rating that can be given); 1.00=onenode (circle of roots), or the equivalent of an entire node, eaten backto within approximately 1½ inches of the stalk (soil line on the 7thnode); 2.00=two complete nodes eaten; 3.00=three or more nodes eaten(highest rating that can be given); and damage in between complete nodeseaten is noted as the percentage of the node missing, i.e. 1.50=1½ nodeseaten, 0.25=¼ of one node eaten, etc.

Results of the field trials against western and northern corn rootwormare shown in Table 8 and against Mexican corn rootworm in Table 9. Alltransgenic corn expressing the FR8a chimeric insecticidal proteinperformed better than a standard commercial chemical insecticide againstwestern, northern and Mexican corn rootworm.

TABLE 8 Results of western and northern corn rootworm field trials.Event Plasmid Root Rating 1 12161 (ubi:FR8a) 0.08 2 12161 0.05 3 121610.09 4 12161 0.04 5 12274 (cmp:FR8a) 0.04 6 12274 0.08 7 12274 0.05Chemical 0.15 Neg Check 0.87

TABLE 9 Results of Mexican corn rootworm field trials. Event PlasmidRoot Rating 1 12161 (ubi:FR8a) 0.04 5 12274 (cmp:FR8a) 0.22 6 12274 0.05Chemical 0.15 Neg Check 1.04

What is claimed is:
 1. A method of making an engineered hybridinsecticidal protein (eHIP), comprising: a. obtaining a Bacillusthuringiensis (Bt) Cry3A protein; b. obtaining a Bt Cry1A or Cry1Abprotein which is different from the Bt Cry3A protein or step a; c.fusing in an N-terminus to C-terminus direction an N-terminal region ofthe Cry3A protein to a C-terminal region of the Cry1Aa or Cry1Abprotein, wherein at least one crossover position between the Cry3Aprotein and the Cry1Aa or Cry1Ab protein is located in conserved block2, conserved block 3 or variable region 4 to make an eHIP that hasactivity against western corn rootworm, wherein the engineeredinsecticidal protein has at least 80% identity to SEQ ID NO: 64; andoptionally d. inserting i.) at the N-terminus a peptidyl fragment; orii.) at the C-terminus a protoxin tail region of a Bt Cry protein, oriii.) both i.) and ii.).
 2. A method of making an engineered hybridinsecticidal protein (eHIP), comprising: a. obtaining a first nucleicacid that encodes a Bacillus thuringiensis (Bt) Cry3A protein; b.obtaining a second nucleic acid that encodes a Bt Cry1Aa or Cry1Abprotein; c. fusing in an 5′ to 3′ direction a 5′ portion of the firstnucleic acid that encodes an N-terminal region of the Cry3A protein to a3′ portion of the second nucleic acid that encodes a C-terminal regionof the Cry1Aa or Cry1Ab protein, wherein at least one crossover positionbetween the first and the second nucleic acids is located in thenucleotides that encode conserved block 2, conserved block 3 or variableregion 4 to make a hybrid nucleic acid that encodes an eHIP that hasactivity against western corn rootworm, wherein the engineeredinsecticidal protein has at least 80% identity to SEQ ID NO:64; andoptionally fusing to the 5′ end of the hybrid nucleic acid a nucleotidesequence that encodes a peptidyl fragment resulting in a 5′ extension orfusing to the 3′ end of the hybrid nucleic acid a nucleotide sequencethat encodes a protoxin tail region of a Bt Cry protein resulting in a3′ extension, or both; d. inserting the hybrid nucleic acid with orwithout one or both of the 5′ or 3′ extensions into an expressioncassette; and e. transforming the expression cassette into a host cell,resulting in the host cell producing an engineered hybrid insecticidalprotein.
 3. The method according to either of claims 1 or 2, wherein theCry3A protein is a Cry3Aa or modified Cry3Aa.
 4. The method according toclaim 3, wherein the engineered hybrid insecticidal protein comprises a.at the C-terminus a protoxin tail region from a Bt Cry protein; or b. atthe N-terminus a peptidyl fragment comprising at least 9 amino acids; orc. both (a) and (b).
 5. The method according to claim 4, wherein theprotoxin tail region is from a Cry1Aa or Cry1Ab.
 6. The method accordingto claim 5, wherein said protoxin tail region comprises at least 38amino acids.
 7. The method according to claim 6, wherein the protoxintail region comprises an amino acid sequence that corresponds to aminoacids 611-648 of SEQ ID NO:
 72. 8. The method according to claim 7,wherein the protoxin tail region comprises amino acids 611-648 of SEQ IDNO:
 72. 9. The method according to claim 4, wherein the peptidylfragment comprises the amino acid sequence YDGRQQHRG (SEQ ID NO: 133) orthe amino acid sequence TSNGRQCAGIRP (SEQ ID NO: 134).
 10. The methodaccording to claim 9, wherein the peptidyl fragment is selected from thegroup consisting of SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQID NO: 129, and SEQ ID NO:
 131. 11. The method of claim 3, wherein (a)the Cry3Aa protein comprises SEQ ID NO:68 or SEQ ID NO: 135, or (b) themodified Cry3Aa comprises SEQ ID NO:70, and the Cry1Ab protein comprisesSEQ ID NO:72.
 12. The method of claim 3, wherein (a) the crossoverposition between Cry3A and Cry1Aa or Cry1Ab is located in a regionbetween amino acids corresponding to amino acid 6 of conserved block 3to amino acid 7 of conserved block 4, or (b) the crossover position islocated is located in conserved block 3 immediately following an aminoacid corresponding to Ser450, Phe454 or Leu468 of SEQ ID NO:70, or (c)the crossover position is located in conserved block 3 immediatelyfollowing Ser450, Phe454 or Leu468 or SEQ ID NO:70.
 13. The method ofclaim 3, wherein the hybrid insecticidal protein comprises at least twocrossover positions between an amino acid sequence from a Cry3A proteinand an amino acid sequence from a Cry1Aa or Cry1Ab protein, wherein (a)the first crossover position between Cry3A and Cry1Aa or Cry1Ab islocated in conserved block 2 immediately following an amino acidcorresponding to Asp232 of SEQ ID NO: 70 and a second crossover positionbetween Cry1Aa or Cry1Ab and Cry3A is located in conserved block 3immediately following an amino acid corresponding to Leu476 of SEQ IDNO: 72; or (b) the first crossover position between Cry3A and Cry1Aa orCry1Ab is located in conserved block 3 immediately following an aminoacid corresponding to Leu468 of SEQ ID NO: 70 and the second crossoverposition between Cry1Aa or Cry1Ab and Cry3A is located in conservedblock 4 immediately following an amino acid corresponding to Ile527 ofSEQ ID NO:
 72. 14. The method of claim 13, wherein the Cry3A is Cry3Aaor modified Cry3Aa and the Cry1A is Cry1Ab, and wherein (a) the firstcrossover position between Cry3Aa and Cry1Ab or modified Cry3Aa andCry1Ab is located in conserved block 2 immediately following Asp232 ofSEQ ID NO: 70 and the second crossover position between Cry1Ab andCry3Aa or modified Cry3Aa is located in conserved block 3 immediatelyfollowing Leu476 of SEQ ID NO: 72; or (b) the first crossover positionbetween Cry3Aa and Cry1Ab or modified Cry3Aa and Cry1Ab is located inconserved block 3 immediately following Leu468 of SEQ ID NO: 70 and thesecond crossover position between Cry1Ab and Cry3Aa or Cry1Ab andmodified Cry3Aa is located in conserved block 4 immediately followingIle527 of SEQ ID NO:
 72. 15. The method of claim 3, wherein theengineered hybrid insecticidal protein comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ IDNO: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 44, SEQ ID NO: 62; SEQID NO: 64, SEQ ID NO: 147, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:159 and SEQ ID NO: 160.